Use of parvovirus capsid particles in the inhibition of cell proliferation and migration

ABSTRACT

The invention described herein relates to the discovery of methods and compositions for the inhibition of growth and/or migration of cells that have a receptor that interacts with a parvovirus B 19  capsid or fragment thereof (e.g., a P antigen containing cell), including but not limited to, cells of hematopoietic origin and endothelial cells. More specifically, parvovirus capsid particles or fragments of parvovirus capsid proteins are used to manufacture medicaments that can be administered to a subject to inhibit hematopoietic progenitor cell growth (e.g., prior to stem cell transplantation), endothelial cell growth, (e.g., as an anti-tumorigenesis treatment or to prevent restenosis or fibrotic build up following prosthetic implantation), or to prevent disorders that involve the abnormal proliferation of cells that have the P antigen (e.g., Polycythemia Vera).

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.09/447,693, filed Nov. 23, 1999, which claims priority to Swedish PatentApplication No. 9804022-3, filed Nov. 24, 1998, both of which are herebyexpressly incorporated by reference in their entireties.

FIELD OF THE INVENTION

[0002] The present invention relates to the discovery of methods andcompositions for the inhibition of cell growth and migration. Morespecifically, B19 parvovirus capsids or fragments of B19 parvoviruscapsid proteins are incorporated into medicaments that can beadministered to a subject to inhibit the growth and/or migration ofcells that have a receptor that interacts with a parvovirus B19 capsidor fragment thereof (e.g., a P antigen containing cell), including, butnot limited to, cells of hematopoietic origin and endothelial cells.

BACKGROUND OF THE INVENTION

[0003] The B19 parvovirus is a human pathogen that can be associatedwith various clinical conditions, ranging from mild symptoms (erythemainfectiosum) to more serious diseases in persons who areimmunocompromised or suffer from hemolytic anemias. Hydrops fetalis andintrauterine fetal death are well-known complications of B19 parvovirusinfection during pregnancy. (Anderson and Young, Monographs in Virology,20 (1997)). The B19 parvovirus particles have icosohedral symmetry, adiameter of 18 to 26 nm, and are composed of 60 capsid proteins,approximately 95% of which are major capsid proteins (VP2) that have amolecular weight of 58 kd. (Fields et al., Virology vol. 2, 3rd edition,Lipponcott-Raven Publishers, Philidelphia, Pa., p. 2202 (1996)).Approximately, 3 -5% of the capsid proteins that compose a B19parvovirus capsid are called minor capsid proteins (VP1), which have amolecular weight of 83 kd, and differ from VP2 by an additional 227amino acids at the amino terminus. (Id.).

[0004] The B19 parvovirus is extraordinarily tropic for human erythroidcells and cultures of bone marrow. B19 parvovirus binds to humanerythroid progenitor cells, for example, and inhibits hematopoieticcolony formation by replicating in these cells. (Brown et al., Science,262:114 (1993) and Mortimer et al., Nature, 302:426 (1983)). Thesuppression of hematopoietic cells has also been seen in bone marrowsamples from infected individuals, resulting in transient anemia and, inrare case, transient pancytopenia. (Saunders et al., Br J Haematol,63:407 (1986)). Further, B19 parvovirus is known to cause bone marrowsuppression in natural and experimental human infections. (Anderson andYoung, Monographs in Virology, 20 (1997)).

[0005] The cellular receptor for B19 parvovirus has been identified asgloboside or erythrocyte P antigen, a textrahexoceramide. (Fields etal., Virology vol. 2, 3rd edition, Lipponcott-Raven Publishers,Philidelphia, Pa., p. 2204 (1996)). The P antigen is found on matureerythrocytes, erythroid progenitors, megakaryocytes, endothelium, kidneycortex, placenta, fetal myocardium (von dem Borne et al., Br J Hematol,63:35 (1986)) and pronormoblasts from fetal liver. (Morey and Flemming,Br J Haematol, 82:302 (1992)). Individuals who genetically lack the Pantigen are not susceptible to B19 parvovirus infection andadministration of either excess P antigen or monoclonal antibodiesdirected to the P antigen can protect erythroid progenitors frominfection with B19 parvovirus. (Id.).

[0006] Additionally, neutralizing antibodies that recognize severalregions of the B19 parvovirus particle have been generated. For example,monoclonal antibodies directed to epitopes of VP2, such as found atamino acids 38-87, 253-272, 309-330, 328-344, 359-382, 449-468, and491-515, and the unique region of VP1 can neutralize B19 parvovirus.(Fields et al., Virology vol. 2, 3rd edition, Lipponcott-RavenPublishers, Philidelphia, Pa., p. 2207 (1996)).

[0007] Genetically engineered expression systems for the production ofB19 parvovirus antigens have also been developed. (Kajigaya et al., ProcNatl Acad Sci USA, 86:7601 (1989); Kajigaya et al., Proc Natl Acad SciUSA, 88:4646 (1991); Brown et al., J Virol, 65:2702 (1991)). Like thenative particles, recombinant B19 parvovirus capsids, produced in abaculovirus system, are composed of both VP1 and VP2 and these capsidproteins self assemble to form virus-like particles (VLPs). (Kajigaya etal., Proc Natl Acad Sci USA, 88:4646 (1991)). Electron microscopicanalyses of the B19 parvovirus capsids revealed that the VLPs arestructurally similar to plasma-derived virions. (Kajigaya et al., ProcNatl Acad Sci USA, 88:4646 (1991)). B19 parvovirus VLPs are currentlybeing evaluated as a potential vaccine against B19 parvovirus infectionand preliminary results show a good neutralizing response without severeside effects. (Bostic et al, J. Infect. Dis., 179:619 (1999). While manyare trying to prevent B19 parvovirus infection by administering B19parvovirus capsids, none have sought to exploit the properties of theB19 parvovirus capsid, B19 parvovirus capsid proteins, or fragmentsthereof to develop novel medicaments that inhibit cell proliferation ormigration.

BRIEF SUMMARY OF THE INVENTION

[0008] It has been discovered that the B19 parvovirus capsid, B19parvovirus capsid proteins, or fragments thereof inhibit the growthand/or migration of cells that have a receptor that interacts with aparvovirus B19 capsid or fragment thereof (e.g., a P antigen containingcell). The data presented herein demonstrate that the B19 parvoviruscapsid, B19 parvovirus capsid proteins, and fragments thereof, inhibithematopoiesis and endothelial cell growth and migration. Accordingly,the compositions described herein can be used to reduce the productionof red blood cells, white blood cells, and blood platelets, as well as,inhibit the growth and migration of other cells having a receptor thatinteracts with a parvovirus B19 capsid or fragment thereof (e.g., a Pantigen containing cell), such as endothelial cells. Because manydiseases or maladies are associated with an overproduction of cells ofhematopoietic origin and/or invasive growth or migration of cells ofendothelial origin, the compositions and methods described herein havesignificant therapeutic and prophylactic utility.

[0009] Embodiments of the invention include medicaments comprising,consisting essentially of, or consisting of B19 parvovirus capsid, B19parvovirus capsid proteins, or fragments thereof, preferably thetripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ. ID. NO. 5), whichcan be administered to subjects in need of an agent that inhibits cellgrowth and/or migration. The B19 parvovirus capsid proteins andfragments thereof, preferably VP2 capsid proteins and fragments thereof,can be prepared synthetically, using peptide chemistry or geneticengineering, or can be made by cleaving B19 parvovirus capsids,desirably VP2 capsids, with various proteases, for example anendoprotease, which cleaves at a lysine or arginine residue.

[0010] The methods of the invention include approaches to manufacturemedicaments that can be used to inhibit the growth or migration of cellsthat have a receptor that interacts with a parvovirus B19 capsid orfragment thereof (e.g., a P antigen containing cell). By one approach,for example, an active ingredient consisting of, consisting essentiallyof, or comprising empty, noninfectious, recombinant B19 parvoviruscapsids, B19 parvovirus capsid proteins, or fragments of B19 parvoviruscapsid proteins, preferably the tripeptide (QQY) and a 10-mer peptide(NKGTQQYTDQ SEQ. ID. NO. 5), are incorporated into a medicament with orwithout a pharmaceutically acceptable carrier or support. The amount ofactive ingredient can be varied depending on the potency of theinhibition needed and the length of treatment period desired from asingle dose.

[0011] The medicaments described herein are preferably used to inhibithematopoietic cell growth, endothelial cell growth, or endothelial cellmigration. The medicaments described herein are used, for example, totreat hematological proliferative disorders, angiogenesis,tumorigenesis, or endothelial cell ingrowth into an implanted prostheticdevice. Further, the medicaments described herein can be provided to asubject, including a fetus, prior to stem cell transplantation.Accordingly, methods of prevention and/or treatment of diseases orconditions associated with hematopoietic or endothelial cellproliferation or migration are aspects of the invention and many of theembodied methods are characterized in that they involve providing asubject at risk of having such a disease or a subject that is afflictedwith such a disease with a therapeutically effective amount of a B19parvovirus capsid, B19 parvovirus capsid proteins, preferably VP2 capsidproteins, or fragments thereof, preferably the tripeptide (QQY) and a10-mer peptide (NKGTQQYTDQ SEQ. ID. NO. 5).

[0012] In other embodiments, methods to prevent or treat PolycytemiaVera are provided, wherein a subject at risk for Polycytemia Vera or asubject afflicted with Polycytemia Vera is identified and said subjectis provided a therapeutically effective amount of a capsid agentselected from the group consisting of B19 parvovirus capsid, B19parvovirus capsid proteins, preferably VP2 capsid proteins, andfragments thereof, preferably the tripeptide (QQY) and a 10-mer peptide(NKGTQQYTDQ SEQ. ID. NO. 5). The capsid agents, described above, can beprovided to said subject neat or as part of a medicament containingother materials including, but not limited to, pharmaceuticallyacceptable supports or carriers. In a similar fashion, otherhematopoietic proliferative disorders can be treated or prevented. Thatis, a subject in need of a capsid agent that inhibits a hematopoieticproliferative disorder is identified and said subject is provided aneffective amount of capsid agent selected from the group consisting ofB19 parvovirus capsid, B19 parvovirus capsid proteins, preferably VP2capsid proteins, and fragments thereof, preferably the tripeptide (QQY)and a 10-mer peptide (NKGTQQYTDQ SEQ. ID. NO. 5). In some embodiments,the progress or effectiveness of treatment is monitored or measured(e.g., analysis of red blood cell hematocrit).

[0013] A method of treating a subject prior to stem cell transplantationis also provided, wherein a subject in need of stem cell transplantationis identified and said subject is provided an effective amount of capsidagent selected from the group consisting of B19 parvovirus capsid, B19parvovirus capsid proteins, preferably VP2 capsid proteins, andfragments thereof, preferably the tripeptide (QQY) and a 10-mer peptide(NKGTQQYTDQ SEQ. ID. NO. 5).

[0014] In more embodiments, methods of inhibiting the growth ormigration of a cell having a receptor that interacts with a parvovirusB19 capsid or fragment thereof (e.g., a P antigen containing cell) areprovided. By one approach, for example, a cell is contacted with acapsid agent selected from the group consisting of B19 parvoviruscapsid, B19 parvovirus capsid proteins, preferably VP2 capsid proteins,and fragments thereof, preferably the tripeptide (QQY) and a 10-merpeptide (NKGTQQYTDQ SEQ. ID. NO. 5), and the inhibition of cell growthor cell migration is monitored or measured. In some embodiments, thecell is of hematopoietic origin or an endothelial cell.

[0015] Methods of inhibiting tissue ingrowth into an implantedprosthesis are also provided. These approaches involve identifying asubject in need of a capsid agent that inhibits tissue ingrowth into animplanted prosthesis and providing said subject an effective amount ofcapsid agent selected from the group consisting of B19 parvoviruscapsid, B19 parvovirus capsid proteins, preferably VP2 capsid proteins,or fragments thereof, preferably the tripeptide (QQY) and a 10-merpeptide (NKGTQQYTDQ SEQ. ID. NO. 5). Approaches to manufacture ingrowthinhibiting implantable prosthesis are also provided, wherein a capsidagent selected from the group consisting of a B19 parvovirus capsid, B19parvovirus capsid proteins, preferably VP2 capsid proteins, andfragments thereof, preferably the tripeptide (QQY) and a 10-mer peptide(NKGTQQYTDQ SEQ. ID. NO. 5) is joined to said implantable prosthesis.Embodiments also include implantable prosthesis, e.g., stents, valves,artificial joints, having said capsid agents.

[0016] More embodiments involve methods of treating or preventingtumorigenesis, wherein a subject in need of a capsid agent that inhibitshematopoietic and/or endothelial cell growth is identified and saidsubject is provided an effective amount of capsid agent selected fromthe group consisting of B19 parvovirus capsid, B19 parvovirus capsidproteins, preferably VP2 capsid proteins, and fragments thereof,preferably the tripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ.ID. NO. 5).

[0017] Still more embodiments concern kits having a capsid agentselected from the group consisting B19 parvovirus capsid, B19 parvoviruscapsid proteins, preferably VP2 capsid proteins, and fragments thereof,preferably the tripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ.ID. NO. 5), and instructions for dosage and administration to a subjectfor hematopoietic progenitor cell growth inhibition, hematopoieticprogenitor cell growth inhibition, endothelial cell growth inhibition ortreatment of a hematological proliferative disease. These kits may alsohave a device to remove blood from a subject and/or a devices to measureblood hematocrit or endothelial cell growth.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1A This figure illustrates the use of parvovirus capsids toinhibit hematopoiesis so as to prevent the overproduction of red bloodcells, white blood cells, and blood platelets.

[0019]FIG. 1B This figure illustrates the general methodology employedin the colony formation assays described herein.

[0020]FIG. 1C This figure shows a graphical representation of theresults of colony formation assays performed on cells from human cordblood that were contacted with varying concentrations of B19 parvoviruscapsids (VP1/2).

[0021]FIG. 2 This figure shows a graphical representation of the resultsof colony formation assays performed on cells from monkey (Baboon andMacaque) bone marrow that were contacted with varying concentrations ofB19 parvovirus capsids (VP1/2).

[0022]FIG. 3 This figure shows a graphical representation of the resultsof colony formation assays performed on cells from human fetal liverthat were contacted with varying concentrations of B19 parvoviruscapsids (BacVP1/2), B19 parvovirus capsids having only VP2 (Bac VP2only), or a control antigen (Bac control antigen).

[0023]FIG. 4 This figure shows a graphical representation of the resultsof colony formation assays performed on cells from fetal liver that werecontacted with varying concentrations of B19 parvovirus VP2 capsids.

[0024]FIG. 5 This figure shows a graphical representation of the resultsof colony formation assays performed on fetal liver cells that werecontacted with varying concentrations of B19 parvovirus VP2 capsids(denoted 8.3.2000) and CPV VP2 capsids (denoted 5).

[0025]FIG. 6 This figure shows a graphical representation of the resultsof colony formation assays performed on fetal liver cells that werecontacted with B19 parvovirus VP2 capsids that had been incubated withdifferent dilutions of anti-B19 parvovirus monoclonal antibody.

[0026]FIG. 7A This figure shows a graphical representation of theresults of colony formation assays performed on fetal liver cells thatwere contacted with intact B19 parvovirus VP2 capsids or fragments ofB19 parvovirus VP2 capsids, which were prepared by digestion of thecapsids with an endoprotease that cleaves at lysine residues.

[0027]FIG. 7B This figure shows a graphical representation of theresults of colony formation assays performed on fetal liver cells thatwere contacted with intact B19 parvovirus VP2 capsids or fragments ofB19 parvovirus VP2 capsids, which were prepared by digestion of thecapsids with an endoprotease that cleaves at arginine residues.

[0028]FIG. 7C This figure shows a graphical representation of theresults of colony formation assays performed on fetal liver cells thatwere contacted with intact B19 parvovirus VP2 capsids or fragments ofB19 parvovirus VP2 capsids, which were prepared by digestion of thecapsids with an endoprotease that cleaves at glutamic acid residues.

[0029]FIG. 8A-H These figures show the results of colony formationassays conducted in the presence of pools of peptides that correspond tooverlapping sequences of the B19 parvovirus VP2 capsid.

[0030]FIG. 9 This figure shows a graphical representation of the resultsof colony formation assays performed on fetal liver cells that werecontacted with a deletion series of peptides that correspond to aP-antigen binding region of the B19 parvovirus VP2 protein.

[0031]FIG. 10 This figure shows a graphical representation of theresults of 11 day colony formation assays performed on fetal liver cellsthat were contacted with the 10 mer NKGTQQYTDQ (SEQ. ID. NO. 5). Twoexperiments are shown (10 mer #1 and 10 mer #2). Colony counts intriplicate are depicted with increasing concentration of peptide plottedas a fraction of colonies formed in medium control (average of 2triplicates=132 colonies). Peptide was added at day 4 in the same amountas at day 0. Error bars=1 SD).

[0032]FIG. 11 This figure shows a bar graph that represents the resultsof cell proliferation assays performed on human umbilical veinendothelial cells (HUVEC) that were contacted with varyingconcentrations of a control antigen (KYVTGIN) (SEQ. ID. NO. 1). On the“x axis” are increasing concentrations of the control antigen (from leftto right), 0 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1.0 μg/ml, and 10.0 μg/ml. Onthe “y axis” are shown spectrophotometric absorbance values taken at 540nm (A₅₄₀).

[0033]FIG. 12 This figure shows a bar graph that represents the resultsof cell proliferation assays performed on human umbilical veinendothelial cells (HUVEC) that were contacted with varyingconcentrations of B19 parvovirus capsids composed of only VP1. On the “xaxis” are increasing concentrations of the B19 parvovirus capsid (VP1)(from left to right), 0 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1.0 μg/ml, and10.0 μg/ml. On the “y axis” are shown spectrophotometric absorbancevalues taken at 540 nm (A₅₄₀).

[0034]FIG. 13 This figure shows a bar graph that represents the resultsof cell proliferation assays performed on human umbilical veinendothelial cells (HUVEC) that were contacted with varyingconcentrations of B19 parvovirus capsid (VP1/2). On the “x axis” areincreasing concentrations of B19 parvovirus capsid (VP1/2) (from left toright), 0 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1.0 μg/ml, and 10.0 μg/ml. Onthe “y axis” are shown spectrophotometric absorbance values taken at 540nm (A₅₄₀).

[0035]FIG. 14 This figure shows a bar graph that represents the resultsof cell proliferation assays performed on human umbilical veinendothelial cells (HUVEC) that were contacted with varyingconcentrations of B19 parvovirus capsid (VP2). On the “x axis” areincreasing concentrations of B19 parvovirus capsid (VP2) (from left toright), 0 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1.0 μg/ml, and 10.0 μg/ml. Onthe “y axis” are shown spectrophotometric absorbance values taken at 540nm (A₅₄₀).

[0036]FIG. 15 This figure shows a bar graph that represents the resultsof cell migration assays performed on human umbilical vein endothelialcells (HUVEC) that were contacted with varying concentrations of B19parvovirus capsids (VP1/2), B19 parvovirus capsids having only VP1 (VP1only), B19 parvovirus capsids having only VP2 (VP2 only), or a controlantigen (control antigen).

DETAILED DESCRIPTION OF THE INVENTION

[0037] It has been discovered that the B19 parvovirus capsid, B19parvovirus capsid proteins, or fragments thereof inhibit the growthand/or migration of cells that have a receptor that interacts with aparvovirus B19 capsid or fragment thereof (e.g., a P antigen containingcell). By using colony formation assays, it was determined that B19parvovirus capsids composed of VP1 and VP2 or VP2 alone inhibithematopoiesis and, thus, the growth of several different types of cellsof hematopoietic origin including human fetal liver cells, humanumbilical cord blood cells, and adult bone marrow cells. Using the sametype of colony formation assay, it was discovered that B19 parvoviruscapsids inhibit the growth of bone marrow cells obtained from Baboonsand Macaques. Further, using colony formation assays, it was discoveredthat fragments of VP2, whether prepared by enzymatic digestion of intactVP2 capsids or by synthesis of peptides corresponding to various regionsof VP2, inhibit hematopoiesis and, thus, the growth of hematopoieticcells. The tripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ. ID.NO. 5) were found to be particularly useful for the inhibition ofhematopoietic cell growth, for example.

[0038] The colony formation assays used in the experiments describedherein are discussed in Ek et al., Bone Marrow Transplantation 11:395-8(1993); Ek et aL , Bone Marrow Transplantation 14:9-14 (1994); Ek etal., Fetal Diagn Therapy 11:318-25 (1996); Ek et al., Fetal DiagnTherapy 11:326-34 (1996); Lindton et al., Fetal Diagn Therapy 15:71-78(2000); Armitage, Blood 92(12):4491-4508 (1998), and Liu et al., Blood90(7) 2583-2590 (1997), all of which are hereby expressly incorporatedby reference in their entireties. As evidenced by the disclosure in theaforementioned references, the in vitro colony formation assays employedherein correlate with and are predictive of in vivo results. In fact,human clinical trials have been predicated on the results from colonyformation assays.

[0039] Through the use of neutralization assays using monoclonalantibodies directed to the P antigen, monoclonal antibodies specific forthe B19 capsid protein, and B19 parvovirus IgG positive sera obtainedfrom two asymptomatic individuals, it was found that B19 parvoviruscapsids inhibit hematopoietic cell growth by interacting with a receptorthat interacts with a parvovirus B19 capsid or fragment thereof (e.g.,the P antigen). Additionally, using immunolabeling, it was discoveredthat the B19 parvovirus capsids were internalized in cells that have areceptor that interacts with a parvovirus B19 capsid or fragment thereof(e.g., a P antigen containing cell).

[0040] It was also discovered that B19 parvovirus capsids inhibit theproliferation and migration of endothelial cells. Endothelial cellproliferation assays were performed by contacting human umbilical veinendothelial cells (HUVEC) with fibroblast growth factor in the presenceof B19 parvovirus capsids. Cell proliferation was monitored by crystalviolet staining and the results established that B19 parvovirus capsidseffectively reduced endothelial cell proliferation. By using a Boydenchamber assay, it was determined that B19 parvovirus capsids inhibitedthe migration of HUVEC cells.

[0041] Several embodiments of the invention involve the manufacture ofmodified B19 parvovirus capsids. Many approaches to manufacture B19parvovirus capsids having less than 5% VP1 and B19 parvovirus capsidshaving only VP2 are disclosed, for example. Further, the manufacture offragments of the B19 parvovirus capsid proteins, and peptidomimeticsresembling these peptides is also disclosed. These fragments can be madesynthetically, by genetic engineering, and by enzymatic digestion ofintact B19 parvovirus capsids. The B19 parvovirus capsids and fragmentsthereof can be used to inhibit the growth and or migration of cells thathave the P antigen.

[0042] The peptide fragments of the invention can be at least 3 aminoacids in length up to and including 780 amino acids in length and cancomprise conservative amino acid substitutions. Additionally, the B19parvovirus capsids, B19 parvovirus capsid proteins, or fragments of B19parvovirus capsid proteins can be modified by the inclusion ofsubstituents that are not naturally found on the B19 parvovirus capsidproteins, the inclusion of mutations, or through the creation of fusionproteins. Derivatized or synthetic B19 parvovirus capsid proteins arealso embodiments.

[0043] Further, approaches to design and manufacture B19 parvoviruscapsids, B19 parvovirus capsid proteins, or fragments of B19 parvoviruscapsid proteins that induce a minimal immune response in a subject so asto allow for the long-term treatment protocols are described. Stillfurther, the construction of profiles on the various B19 capsid-basedtherapeutics, which includes information such as sequences, sites ofmutations or modifications, performance information in functionalassays, and therapeutic information including disease indications,clinical evaluations and the like are embodiments of the invention.

[0044] Other embodiments of the invention include multimeric agentscontaining B19 parvovirus capsids, B19 parvovirus capsid proteins, orfragments of B19 parvovirus capsid proteins, including, but not limitedto, the tripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ. ID. NO.5) and methods of making these compositions. These multimeric agents(collectively referred to as “capsid agents”) are created by joining B19parvovirus capsids, B19 parvovirus capsid proteins, or fragments of B19parvovirus capsid proteins to a support, which can be a bead, a resin, aplastic dish, and, preferably, a medical device, such as a stent, valve,or other prosthetic. In some embodiments, the capsid agents provide apotent inhibitor of the proliferation and/or migration of cells thathave the P antigen (e.g., restenosis following implantation). Thesemultimeric capsid agents can be used to inhibit cell growth andmigration and also can be used to isolate cells having the P antigen(e.g., affinity chromatography).

[0045] The preparation of many different pharmaceuticals and medicaldevices that comprise B19 parvovirus capsids, B19 parvovirus capsidproteins, or fragments of B19 parvovirus capsid proteins is alsodescribed herein. These devices are made by joining B19 parvoviruscapsids, B19 parvovirus capsid proteins, or fragments of B19 parvoviruscapsid proteins or capsid agents directly or indirectly (e.g., through alinker or support) to said devices. These pharmaceuticals andmedicaments can be formulated with other additives, carriers, orexcipients so as to allow administration by many routes.

[0046] Therapeutic and prophylactic methods are also described. Somemethods, for example, involve approaches to inhibit hematopoiesis or theproliferation and/or migration of cells that have the P antigen,including, but not limited to cells of hematopoietic origin andendothelial cells. These methods are practiced by administering atherapeutic comprising B19 parvovirus capsids, B19 parvovirus capsidproteins, or fragments of B19 parvovirus capsid proteins, preferably thetripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ. ID. NO. 5).

[0047] For example, a method to prevent or treat Polycytemia Vera isprovided, wherein a subject at risk for Polycytemia Vera or a subjectafflicted with Polycytemia Vera is identified and the subject isprovided a therapeutically effective amount of B19 parvovirus capsid,B19 parvovirus capsid proteins, preferably VP2 capsid proteins, andfragments thereof, preferably the tripeptide (QQY) or a 10-mer peptide(NKGTQQYTDQ SEQ. ID. NO. 5). In similar methods, other hematopoieticproliferative disorders can be treated or prevented. That is, a subjectin need of a capsid agent that inhibits a hematopoietic proliferativedisorder is identified and said subject is provided an effective amountof B19 parvovirus capsid, B19 parvovirus capsid proteins, preferably VP2capsid proteins, or fragments thereof, preferably the tripeptide (QQY)and a 10-mer peptide (NKGTQQYTDQ SEQ. ID. NO. 5). In some embodiments,the progress or effectiveness of treatment is monitored or measured(e.g., analysis of red blood cell hematocrit).

[0048] Embodiments of the invention also concern methods to inhibithematopoiesis in a subject prior to in utero stem cell transplantation.These methods are practiced by providing to a subject in need of stemcell transplantation a B19 parvovirus capsids, B19 parvovirus capsidproteins, or fragments of B19 parvovirus capsid proteins, preferably thetripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ. ID. NO. 5). In arelated method, hematopoiesis is inhibited in a subject prior to postnatal stem cell transplantation (e.g., a novel approach tonon-myeloblative therapy) by providing said subject B19 parvoviruscapsids, B19 parvovirus capsid proteins, or fragments of B19 parvoviruscapsid proteins, preferably the tripeptide (QQY) and a 10-mer peptide(NKGTQQYTDQ SEQ. ID. NO. 5).

[0049] Still more embodiments involve methods of preventingangiogenesis, tumorigenesis, or cancer and methods of preparing medicaldevices, such as stents or valves, that prevent fibrotic build up orrestenosis or otherwise delay endothelial cell ingrowth. These methodsare also practiced by providing B19 parvovirus capsids, B19 parvoviruscapsid proteins, or fragments of B19 parvovirus capsid proteins,preferably the tripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ.ID. NO. 5) to a subject at risk of angiogenesis, tumorigenesis, orcancer or to a medical device such as a stent or valve.

[0050] Kits comprising B19 parvovirus capsids, B19 parvovirus capsidproteins, or fragments of B19 parvovirus capsid proteins preferably thetripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ. ID. NO. 5) arealso embodiments of the invention. These kits may contain instructionsrelating to dosage and time of administration. These kits may also havea device to remove blood from a subject and/or a device to measure bloodhematocrit or endothelial cell growth. The section below describes theexperiments that provided evidence that B19 parvovirus capsids inhibitthe growth of hematopoietic cells.

[0051] B19 parvovirus capsids inhibit hematopoiesis and the growth ofhematopoietic cells In a first set of experiments, it was discoveredthat B19 parvovirus capsids inhibit hematopoiesis and that thesemolecules could be used to reduce the overproduction of red blood cells,white blood cells, and blood platelets, as is experienced by patientssuffering from hematological proliferative diseases, such as PolycytemiaVera. (See FIG. 1A). In a first set of experiments, fresh human fetalliver cells, umbilical cord blood cells, and bone marrow cells werecontacted with recombinant B19 parvovirus capsids in a colony formationassay. The general features of a colony formation assay are depicted inFIG. 1B. The ability of the B19 parvovirus capsids to inhibithematopoiesis was verified by the reduction of colony formation in thepresence of the capsids, as compared to untreated control populations ofcells. A more detailed description of these experiments is providedbelow.

[0052] To obtain fetal liver tissue, human fetuses 6-12 weeks ofgestational age were obtained from legal abortions; the patients hadvolunteered to donate fetal tissue. Gestational age was estimatedaccording to specific anatomical markers and is given as menstruationalage. Abortions were performed with vacuum aspirations. Fetal liver wasdissected under sterile conditions, placed in a sterile tube containingRPMI1640 and disintegrated by passage through a vinyl mesh to form asingle cell suspension. Nucleated cells were then washed three times,counted and diluted in culture medium.

[0053] A commercial kit—the “Stem cell CFU kit” (GIBCO BRL, LifeTechnology Inc., NY, USA)—was used to perform the colony formationassays. The kit provides a semi-solid support that mimics theextracellular matrix produced by stromal cells. Other componentsincluded in the kit are: Iscove's modified Dulbecco's medium, modifiedfetal bovine serum, methylcellulose, 2-mercaptoethanol, conditionedmedium and erythropoietin. The colonies that were formed in the assaywere identified as BFU-E (burst forming unit-erythroid cells) withdensely packed hemoglobonized cells, CFU-GM (colony formingunits-granulocytes, macrophages) with arrangements of non-hemoglobinizedcells, and CFU-GEMM (colony forming units-granulocytes, erythroid cells,macrophages, megakaryocytes) with hemoglobinized cells and small andlarge peripheral cells.

[0054] Recombinant B19 parvovirus empty capsid particles (Kajigaya etal., Proc Natl Acad Sci USA, 88:4646 (1991)) were prepared in arecombinant baculovirus-insect cell (Spodofera frugiperda) expressionsystem. (Kajigaya et al., Proc Natl Acad Sci USA, 88:4646 (1991)). Theisolated or purified capsids were diluted in buffer (20 mM Tris, 0.5MNaCl, pH 8.5) and 30 μL of each dilution was added to approximately25×10³ cells (50×10³ for postnatal cells) in 100 μL of culture mediumand incubated for 1 hour at 4° C. The mixtures were then transferred toincubation dishes and culture medium was added to a final volume of 0.5ml per well. The cells were then incubated for 11 days in a humidifiedatmosphere at 5% CO₂, and were scored for BFU-E, CFU-GM and CFU-GEMMderived colony formation.

[0055] In the 11-day colony formation assay, described above, it wasfound that B19 parvovirus capsids inhibited hematopoietic cell growth,as evidenced by a reduction in colony formation of fresh human fetalliver cells, umbilical cord blood cells, and adult bone marrow cells.That is, a reduction in colony formation of BFU-E (burst formingunit-erythroid), CFU-GM (colony forming unit-granulocyte, macrophage)and CFU-GEMM (colony forming unit-granulocyte, erythrocyte, monocyte,megakaryocyte) cells was observed when human fetal liver cells,umbilical cord blood cells, and adult bone marrow cells were incubatedwith the B19 parvovirus capsids. (See Table 1). TABLE 1 Colony-formingunit assay of fetal liver cells*. Dilution of B19 parvovirus ColonyCounts (% of medium control) capsid (μg/ml) BFU-E CFU-GM CFU-GEMM 70.022% 14% 31%  0.7 39% 54% 63%  0.007 79% 95% 94% Medium (= 100%), counts95    37    16   

[0056] As shown in Table 1, an inhibition of the colony formation ofhematopoietic cells was seen with as little as 0.007μg/ml B19 parvoviruscapsid and considerable inhibition was observed at 70.0 μg/ml B19parvovirus capsid. Recombinant papillomavirus capsids (Cottontail rabbitpapillomavirus and human papillomavirus type 6) were included in thecolony formation assays as controls. These control capsids arestructurally similar to parvovirus capsids but do not interact with theP antigen. The control capsids (tested in the range 0.01-100 μg/ml) hadno effect on colony formation.

[0057] In other experiments, it was found that the colony formation ofhematopoietic cells could be rescued by incubating B19 parvoviruscapsids with anti-B19 parvovirus monoclonal antibodies or with B19parvovirus IgG positive human sera, prior to adding the mixture to thecells. The anti-B19 parvovirus monoclonal antibody (MAB8292), which isan IgG class antibody, was purchased (Chemicon AB, Malmo, Sweden) andthe B19 parvovirus IgG positive (B19 parvovirus IgM negative) sera wereobtained from two asymptomatic individuals. In one neutralizationexperiment, B19 parvovirus capsids were incubated with anti-B19parvovirus monoclonal antibody (MAB8292) prior to adding the mixture tofetal liver cells. Approximately, 25 μl of anti-B19 parvovirusmonoclonal antibody (MAB8292) was incubated with 25 μl of B19 parvoviruscapsids for 2 hours at 4° C. The mixtures were then added to the cellsand the 11-day colony formation assay, as described above, was performedon the “neutralized”-capsid/cell mixture.

[0058] Although a relatively high concentration of B19 parvoviruscapsids was used (7 μg/ml, as compared to the values in Table 1), aslittle as 0.02 μg/ml of the anti-B19 monoclonal antibody reduced theability of B19 parvovirus capsids to inhibit colony formation and aconcentration of 20.0 μg/ml of the anti-B19 parvovirus monoclonalantibody completely blocked the inhibition on BFU-E colony formation anddrastically reduced the effect on CFU-GM and CFU-GEMM colony formation(See Table 2). TABLE 2 Neutralization assay using anti-B19 parvovirusmonoclonal antibody*. B19 parvovirus capsid (7 μg/ml) + Colony Countsdilutions of Anti-B19 parvovirus (% of medium control) Mab (μg/mL) BFU-ECFU-GM CFU-GEMM 20.0 >100%    74% 67%  2.0 69% 35% 45%  0.2 52% 21% 19% 0.02 52% 30% 21% capsid only 43% 30% 21% Medium (= 100%), counts114     66    42   

[0059] Similarly, the two lots of B19 parvovirus IgG positive sera wereanalyzed for their ability to neutralize the B19 parvovirus capsids.Approximately, 25 μl of B19 parvovirus IgG positive serum was incubatedwith 25 μl of B19 parvovirus capsids for 2 hours at 4° C., then themixtures were added to fetal liver cells. Subsequently, the colonyformation assay described above was performed on the sera-neutralizedB19 parvovirus capsid/cell mixture. As shown in Table 3, in the absenceof sera, B19 parvovirus capsids (0.14 μg/ml) significantly inhibitedfetal liver cell colony formation, whereas, as little as a 1:100dilution of serum 1 reduced the ability of B19 parvovirus capsids toinhibit fetal liver cell growth. TABLE 3 Neutralization assay usinghuman B19 parvovirus IgG positive sera*. B19 parvovirus capsid (0.14μg/ml) + Colony Counts dilutions of two human (% of medium control) B19parvovirus IgG positive sera BFU-E CFU-GM CFU-GEMM Serum 1, 1:10 70% 78%90% Serum 1, 1:100 25% 23% 40% Serum 2, 1:10 48% 57% 57% Serum 2, 1:10017% 27% 67% capsid only 18% 17% 63% Medium (= 100%), counts 157    81    30   

[0060] Further evidence that B19 parvovirus capsids inhibit the colonyformation of hematopoietic cells was obtained by performingneutralization assays using monoclonal antibodies directed to theP-antigen. The anti-P monoclonal antibody (CLB-ery-2), is a mouse IgMclass antibody. (See von dem Borne et al., Br J Hematol, 63:35 (1986),herein expressly incorporated by reference in its entirety). In theseassays, approximately 2.5×10⁴ fetal liver cells were suspended in 100 μIof medium and were then incubated with either 25 μl of anti-P monoclonalantibody (CLB-ery-2) or 25 μl of anti-PI (Seraclone), a controlmonoclonal antibody. Cells and monoclonal antibody were incubated for 1hour at 4° C. The cell/antibody mixtures were washed twice in coldculture medium prior to adding the B19 parvovirus capsids and,subsequently, the colony formation assays were conducted, as previouslydescribed.

[0061] In accord with the evidence from the previous experiments andstudies conducted with native viral particles (See Brown et al.,Science, 262:114 (1993)), it was discovered that the monoclonalantibodies directed to the P antigen could restore growth of fresh fetalliver cells incubated in the presence of B19 parvovirus capsids. Asshown in Table 4, the inhibitory effect of the B19 parvovirus capsid wasreduced by at least 25% when the cells were incubated in the presence ofCLB-ery-2. In contrast, the anti-P₁ (Seraclone) monoclonal antibody(Labdesign, Stockholm, Sweden), which does not interact with the Pantigen, had no effect on colony formation as compared to the B19parvovirus capsid control. TABLE 4 Neutralization assay using anti-P orantiP₁ monoclonal antibodies*. Colony Counts (% of medium control) BFU-ECFU-GM CFU-GEMM B19 parvovirus capsid (0.14 μg/ml) + of Anti-P Mab(titer) 1:5 51% 39% 93% 1:500 23% 10% 43% capsid only 18% 17% 63% Medium(= 100%), counts 157     81    30    B19 parvovirus capsid (0.14μg/mL) + of Anti-P₁ Mab (μg/ml) 400.0 25% 20% 50%  4.0 17% 22% 47%capsid only 18% 17% 63% Medium (= 100%), counts 157     81    30   

[0062] The inhibitory effect of B19 parvovirus capsids on colonyformation was also tested using fresh stem cells derived from cord bloodand adult bone marrow samples. Colony formation assays in the presenceof B19 parvovirus capsids were performed on cells obtained fromumbilical cord blood and bone marrow using the protocol described above.Umbilical cord blood samples were obtained immediately after vaginaldelivery from normal births. Samples of adult bone marrow were obtainedfrom healthy allogeneic donors. Suspensions of fresh cells wereheparinized and diluted in 0.9% NaCl and separated on Lymphoprep(Nycomed, Parma, Oslo, Norway) for gradient centrifugation at 2000 rpmfor 20 min. Cells were carefully removed with a Pasteur pipette, washedthree times in 0.9% NaCl, counted and diluted in culture medium inpreparation for the colony formation assays.

[0063] The ability of B19 parvovirus capsids to inhibit hematopoieticcells obtained from cord blood and bone marrow was comparable to thatexhibited with fetal liver cells (See Table 5). As shown in FIG. 1C, forexample, the growth of cells obtained from human cord blood decreased asthe concentration of B19 parvovirus capsid increased. Further,neutralization assays using cells obtained from cord blood or bonemarrow and B19 parvovirus capsids also exhibited results similar tothose seen with human fetal liver cells. That is, B19 parvovirus capsidsthat were incubated with the anti-B19 parvovirus monoclonal antibody(Mab8292) prior to contact with the cells obtained from cord blood andbone marrow demonstrated a reduced ability to inhibit cell growth, asevidenced by an increase in colony formation. TABLE 5 Colony formationassay on cord blood and adult bone marrow cells Colony Counts (% ofmedium control) B19 parvovirus capsid (μg/ml) BFU-E CFU-GM CFU-GEMM Cordblood cells 7.0 10% 54% 43% 0.7 33% 62% 43% 0.07 49% 72% 50% 0.007 57%67% 70% 0.0007 84% 79% 93% Medium (= 100%), counts 134     39    30   Bone marrow cells 7.0 18% 36%  6% 0.7 43% 45% 28% 0.07 63% 41% 44% 0.00776% 80% 78% 0.0007 86% 77% 78% Medium (= 100%), counts 134     39   30   

[0064] Additionally, colony formation assays in the presence of B19parvovirus cells were performed, as described above, using hematopoieticcells obtained from the bone marrow of monkeys (Baboons and Macaques).As shown in FIG. 2, primate hematopoietic cell growth decreased inconcordance with an increase in concentration of B19 parvovirus capsid.The results from this experiment not only demonstrate that primatehematopoietic cells have a receptor that interacts with B19 parvoviruscapsids but also established that Baboon and Macaque primates aresuitable for in vivo study of the therapeutic and prophylacticembodiments of the invention.

[0065] In an effort to identify the regions of the B19 parvovirus capsidthat are involved in inhibiting cell growth, it was observed that afterbinding, the capsid fuses with cells having the P antigen and thenbecomes internalized. In one experiment that provided evidence of B19parvovirus capsid internalization, fetal liver cells were incubated withB19 parvovirus capsids and the capsid treated cells were fixed on BioRadslides, labeled with the anti-B19 parvovirus monoclonal antibody(Mab8292), and detected with a fluorescent secondary antibody. By thisapproach, fetal liver cells were washed in PBS and a suspension with acell concentration of approximately 2×10⁶/ml was prepared. A fraction ofthe suspension was incubated with B19 parvovirus native capsid, (0.35 μgcapsid/ml cell suspension), in 37° C. for 1 hour. Approximately, 20 μldroplets (about 40,000 cells) of cell/capsid suspension was then placedon two BioRad slides, 10 wells on each slide. In two of the wells oneach slide, cells that had not been treated with capsids were used ascontrols.

[0066] Next, the cells on one of the two BioRad® slides werepermeablized with saponine, which promotes antibody penetration.Subsequently, primary anti-B19 parvovirus monoclonal IgG antibody wasadded and, after binding and removal of unbound primary antibody with aPBS wash, the secondary fluorescent anti-IgG antibody was added, allowedto bind, and the unbound secondary antibody was removed with a PBS wash.A UV-light microscope was used for the analysis. Saponin permeablizedcells treated with B19 parvovirus capsids exhibited fluorescence on cellmembranes and inside the cells. In contrast, control cells, which werenot permeablized with saponin, exhibited fluorescence only at the cellsurface. These results provided evidence that the inhibition of cellgrowth mediated by the B19 parvovirus capsid may involve more than areceptor/ligand interaction. The next section describes the discoverythat modified B19 parvovirus capsids can be used to inhibithematopoiesis and/or hematopoietic cell growth.

[0067] Modified B19 parvovirus capsids inhibit hemaptopoiesis andhematopoietic cell growth

[0068] Although some embodiments of the invention comprise B19parvovirus capsids without modification, native B19 parvovirus VLPs(i.e., capsids having 95% VP2 and 5%VP1) elicit an immune response,which makes them less desirable for some therapeutic applications (e.g.,use in long term treatment protocols), others have constructed amodified B19 parvovirus capsid having 25% VP1 and 75% VP2 while tryingto develop a parvovirus vaccine, however, this modified VLP induces anelevated neutralizing response in vivo. (See U.S. Pat. No. 5,508,186 toYoung et al., herein expressly incorporated by reference in itsentirety). Such modified B19 parvovirus capsids are undesirable forlong-term therapeutic protocols because a subject's immune response canquickly clear the VLPs from the subject's body, thus, lowering theeffective dose. Additionally, since the seroprevalence of antibodies toparvovirus in the population approaches 50-70%, it is preferred thattreatment protocols use capsid agents that elicit a minimal immuneresponse.

[0069] Since the unique region of VP1 appears to play an integral rolein immune response to the B19 parvovirus capsid (See Fields et al.,Virology vol. 2, 3rd edition, Lipponcott-Raven Publishers, Philidelphia,Pa., p. 2207 (1996)), modified capsids that comprise less VP1 than isfound in nature can be manufactured and can be more effectivetherapeutics for long term use. Thus, some embodiments include B19parvovirus capsids that comprise, consist essentially of, or consist ofan amount of VP1 that is less than or equal to about 0.1% to about 5.0%.That is, the amount of VP1 can be about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%,1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7, 2.8%, 2.9%,3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%,4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, and 5.0% of the totalamount of VP1 and VP2.

[0070] Further, recombinant VP2 can spontaneously form capsid structuresthat are similar to the VP1/VP2 structure without VP1. (See U.S. Pat.No. 5,508,186 to Young et al.). The VP2 capsids have only minorneutralizing regions and, thus, can be very effective therapeutics foruse in long-term treatment protocols. Thus, some embodiments include B19parvovirus capsids that comprise, consist essentially of, or consist ofan amount of VP2 that is about 95% or more; that is, more than or equalto 100%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, and 95%of the total amount of VP1 and VP2.

[0071] By employing the colony formation assays described above, it wasdiscovered that VP2 capsids can also inhibit hematopoiesis and thegrowth of hematopoietic cells. (See FIG. 3). As above, human fetal livercells were incubated in the presence of the VP2 capsids and the 11 daycolony formation assay was performed. The positive control in theseexperiments was the native B19 parvovirus VLP, that is, the B19parvovirus capsids having 95% VP2 and 5% VP1 (VP1/2). The results shownin FIG. 3 verify that the VP2 capsids inhibit hematopoietic cell growthat concentrations as low as 3μg/ml and significant inhibition occurs at30μg/ml.

[0072] In a similar experiment, it was determined that B19 parvovirusVP2 capsids (8.3.2000 VP2) inhibited colony formation of hematopoieticstem cells as effectively as VP1/2 capsids (See FIG. 4). Approximately50% inhibition was seen at 1 μg/ml VP2 capsid, whereas approximately 40%inhibition (60% of the medium control) was seen at 0.07μg/ml VP1/2capsid. (See FIG. 3). In these experiments, fetal liver cells wereincubated with dilutions of the B19 parvovirus capsids prior to the11-day colony formation assay. Data are plotted in comparison with themedium control (the medium control being 1.0) and represent fourdifferent experiments, each one in triplicate (mean +/−1 SD).

[0073] Recombinant VP2 capsid proteins derived from a canine parvovirus(CPV) strain (denoted 5) did not inhibit colony formation in assayswhere VP2 capsids (8.3.2000) reduced colony formation to less than 30%of the control. (See FIG. 5). The effect of B19 parvovirus VP2 capsids(“8.3.2000”, 30 μg/mL) and CPV (canine parvovirus) VP2 capsids (“5”, 30μg/mL) in a colony-forming unit assay of fetal liver cells is shown inFIG. 5. The cells were incubated with the capsids prior to the 11-daycolony formation assay. Data are plotted in comparison with the mediumcontrol (the medium control being 1.0) and represent two differentexperiments (gray and black bars, respectively), each one in triplicate(mean+/−1 SD). The CPV capsids were baculovirus-produced (in Sf-9 cellcultures) and were purified by ultracentrifugation for one hour. Theresults above provided strong evidence that B19 VP2 capsids inhibithematopoiesis and the proliferation of cells having the P antigen.

[0074] To verify that the B19 parvovirus VP2 capsids were, in fact, theagent responsible for the inhibition of cell growth, neutralizationassays, as described above, were conducted. As described for the B19parvovirus VP1/VP2 constructs, Lindton et al., Fetal Diagn. Ther.16(1):26-31 (2001), herein expressly incorporated by reference in itsentirety, the inhibitory effect of the VP2 protein on colony formationwas partially neutralized by a monoclonal antibody directed to theVP1/VP2 protein (See FIG. 6). In these experiments, B19 parvovirus VP2capsids were incubated with different dilutions of an anti-B19parvovirus monoclonal antibody (MAB 8292 obtained from Chemicon) andwere then incubated with fetal liver cells prior to the colony-formationassay. The data in FIG. 6 are plotted in comparison with the mediumcontrol (the medium control being 1.0) and represent four differentexperiments, each one in triplicate (mean+/−1 SD). Two donors of fetalliver cells were used for comparison (#1 and #2, respectively). Althougha total neutralizing effect was not reached, which may be due to thefact that the monoclonal antibody is not specific to the VP2 proteinalone, the data from this experiment verified the inhibition of colonyformation mediated by the B19 parvovirus VP2 capsids. The section belowdescribes the discovery that fragments of the B19 parvovirus VP2 capsidinhibit hematopoiesis and the growth of cells that have the P antigen.

[0075] Fragments of B19 parvovirus VP2 capsids inhibit hemaptopoiesisand hematopoietic cell growth

[0076] In addition to intact B19 parvovirus VP2 capsids, fragments ofB19 parvovirus VP2 capsids can be used to inhibit hematopoiesis and thegrowth of P antigen containing cells. In another set of experiments, B19parvovirus VP2 capsids were enzymatically cleaved and the resultingcleavage products were found to inhibit hematopoiesis in colonyformation assays. (See FIGS. 7A-C). In these experiments, B19 parvovirusVP2 capsids were digested with three different endoproteases: LYS-Cendoprotease sequencing grade; ARG-C endoprotease sequencing grade; andGLU-C endoprotease sequencing grade obtained from Roche. The cleavageproducts were contacted with fetal liver cells, which were thensubjected to a colony formation assay. That is, the cells were incubatedwith various dilutions of the B19 parvovirus VP2 fragments prior to the11-day colony formation assay, as described above. Data are plotted as apercentage of the medium control (the medium control being 1.0) andrepresent one experiment, performed in triplicate (mean+/−1 SD). Silverstaining of a gel in which the cleavage products were separatedconfirmed that no intact B19 parvovirus VP2 capsid remained. The datashow that the cleavage products created by digestion with anendoprotease that cleaves at either Lysine residues or Arginine residuesbut not at Glutamic acid residues are fragments of B19 parvovirus VP2capsid protein that effectively inhibit colony formation. Additionally,the data showed that specific fragments or regions of the VP2 proteinmay be involved in producing the inhibitory effect.

[0077] Overlapping peptides (20mers) encompassing the entire B19parvovirus VP2 protein (with a 10 amino acid overlap) were synthesized.These peptides were grouped into eight different pools (pools 1-7containing 7 peptides and pool 8 containing 6 peptides) and each pool ofpeptides was tested for the ability to inhibit hematopoiesis andhematopoietic cell growth in colony formation assays. (See FIGS. 8A-H).In these experiments, the cells were incubated with various dilutions ofthe peptides prior to the 11-day colony formation assay. Each pool ofpeptides showed some degree of inhibition of colony formation and thepeptides of pool VI showed significant inhibition. (See FIG. 8F). Thesequences of the peptides of each pool are provided in Table 6. TABLE 6VP2 peptide pools* POOL 1   1 (position in protein) MTSVNSAEASTGAGGGGSNP(SEQ ID NO.9)           TGAGGGGSNPVKSMWSEGAT (SEQ ID NO.10)                    VKSMWSEGATFSANSVTCTF (SEQ ID NO.11)                              FSANSVTCTFSRQFLIPYDP (SEQ ID NO.12)                                        SRQFLIPYDPEHHYKVFSPA (SEQ IDNO.13)                                                  EHHYKVFSPAASSCHNASGK(SEQ ID NO.14)                                                          ASSCHNASGKEAKVCTISPI(SEQ ID NO.15) POOL 2  71 EAKVCTISPIMGYSTPWRYL (SEQ ID NO.16)          MGYSTPWRYLDFNALNLFFS (SEQ ID NO.17)                    DFNALNLFFSPLEFQHLIEN (SEQ ID NO.18)                              PLEFQHLIENYGSIAPDALT (SEQ ID NO.19)                                        YGSIAPDALTVTISEIAVKD (SEQ IDNO.20)                                                  VTISEIAVKDVTDKTGGGVQ(SEQ ID NO.21)                                                             VTDKTGGGVQVTDSTTGRLC(SEQ ID NO.22) POOL 3 141 VTDSTTGRLCMLVDHEYKYP (SEQ ID NO.23)          MLVDHEYKYPYVLGQGQDTL (SEQ ID NO.24)                    YVLGQGQDTLAPELPIWVYF (SEQ ID NO.25)                              APELPIWVYFPPQYAYLTVG (SEQ ID NO.26)                                        PPQYAYLTVGDVNTQGISGD (SEQ IDNO.27)                                                  DVNTQGISGDSKKLASEESA(SEQ ID NO.28)                                                             SKKLASEESAFYVLEHSSFQ(SEQ ID NO.29) POOL 4 211 FYVLEHSSFQLLGTGGTATM (SEQ ID NO.30)          LLGTGGTATMSYKFPPVPPE (SEQ ID NO.31)                    SYKFPPVPPENLEGCSQHFY (SEQ ID NO.32)                              NLEGCSQHFYEMYNPLYGSR (SEQ ID NO.33)                                        EMYNPLYGSRLGVPDTLGGD (SEQ IDNO.34)                                                  LGVPDTLGGDPKFRSLTHED(SEQ ID NO.35)                                                             PKFRSLTHEDHAIQPQNFMP(SEQ ID NO.36) POOL 5 281 HAIQPQNFMPGPLVNSVSTK (SEQ ID NO.37)          GPLVNSVSTKEGDSSNTGAG (SEQ ID NO.38)                    EGDSSNTGAGKALTGLSTGT (SEQ ID NO.39)                              KALTSLSTGTSQNTRISLRP (SEQ ID NO.40)                                        SQNTRISLRPGPVSQPYHHW (SEQ IDNO.41)                                                  GPVSQPYHHWDTDKYVTGIN(SEQ ID NO.42)                                                             DTDKYVTGINAISHGQTTYG(SEQ ID NO.43) POOL 6 351 AISHGQTTYGNAEDKEYQQG (SEQ ID NO.44)          NAEDKEYQQGVGRFPNEKEQ (SEQ ID NO.45)                    VGRFPNEKEQLKQLQGLNMH (SEQ ID NO.46)                              LKQLQGLNMHTYFPNKGTQQ (SEQ ID NO.47)                                        TYFPNKGTQQYTDQIERPLM (SEQ IDNO.48)                                                  YTDQIERPLMVGSVWNRRAL(SEQ ID NO.49)                                                             VGSVWNRRALHYESQLWSKI(SEQ ID NO.50) POOL 7 421 HYESQLWSKIPNLDDSFKTQ (SEQ ID NO.51)          PNLDDSFKTQFAALGGWGLH (SEQ ID NO.52)                    FAALGGWGLHQPPPQIFLKI (SEQ ID NO.53)                              QPPPQIFLKILPQSGPIGGI (SEQ ID NO.54)                                        LPQSGPIGGIKSMGITTLVQ (SEQ IDNO.55)                                                  KSMGITTLVQYAVGIMTVTM(SEQ ID NO.56)                                                             YAVGIMTVTMTFKLGPRKAT(SEQ ID NO.57) POOL 8 491 TFKLGPRKATGRWNPQPGVY (SEQ ID NO.58)          GRWNPQPGVYPPHAAGHLPY (SEQ ID NO.59)                    PPHAAGHLPYVLYDPTATDA (SEQ ID NO.60)                              VLYDPTATDAKQHHRHGYEK (SEQ ID NO.61)                                        KQHHRHGYEKPEELWTAKSR (SEQ IDNO.62)                                                   PEELWTAKSRVHPL(SEQ ID NO.63)

[0078] Additionally, peptides corresponding to regions of the B19parvovirus VP2 capsid believed to be involved in binding to the Pantigen were synthesised and analysed for their ability to inhibithematopoiesis and the proliferation of hematopoietic cells. Chipman etal., Proc Natl Acad Sci USA 93:7502-7506 (1996), herein expresslyincorporated by reference in its entirety. (See Table 7). Table 7 showsa deletion series of synthetic peptides that were screened to determinetheir ability to inhibit hematopoesis and cell growth. (See FIG. 9).TABLE 7 Deletion series of P antigen binding region of parvovirus B19VP2 protein* Peptide I: GLNMHTYFPNKGTQQYTDQIE (SEQ ID NO:2) Peptide II:     TYFPNKGTQQYTDQIE (SEQ ID NO:3) Peptide III:          NKGTQQYTDQIE(SEQ ID NO:4) Peptide IV:           NKGTQQYTDQ (SEQ ID NO:5) Peptide V:          NKGTQQYT (SEQ ID NO:6) Peptide VI:               QQYTDQ (SEQID NO:7) Peptide VII:               QQYQ (SEQ ID NO:8) Peptide VIII:               QQY

[0079] Among the peptides in Table 7, peptides IV-VIII showed goodinhibition and the 3-mer (QQY) and the 10-mer (NKGTQQYTDQ SEQ ID NO. 5)were found to significantly inhibit hematopoiesis and cell growth. (SeeFIG. 9). Data are plotted in comparison with the medium control (themedium being 1.0) and represent one experiment in triplicate (mean+/−1SD). As shown in FIG. 10, the 10-mer peptide showed significantinhibition of colony formation at about 1 to about 100 μM concentration.The next section describes the discovery that B19 parvovirus capsids caninhibit another type of cell that has a receptor that interacts with aparvovirus B19 capsid or fragment thereof—the endothelial cell.

[0080] B19 parvovirus capsids inhibit the growth of other cells thatexpress a receptor that interacts with a parvovirus B19 capsid orfragment thereof including, but not limited to, endothelial cells

[0081] The results from the experiments described in the precedingsections provide evidence that B19 parvovirus capsids, B19 parvovirusVP2 capsids, and fragments of B19 parvovirus VP2 capsids efficientlyinhibit the growth of a number of different hematopoietic cells thathave a receptor that interacts with a parvovirus B19 capsid or fragmentthereof (e.g., a P antigen containing cell), including hematopoieticcells from different species. In a another set of experiments, it wasdiscovered that recombinant B19 parvovirus capsids inhibit the growth ofendothelial cells, as evidenced by a reduction in endothelial cellproliferation and migration. A description of these experiments isprovided below.

[0082] To determine whether B19 parvovirus capsids can inhibitendothelial cell proliferation, assays were performed in which primaryhuman umbilical vein endothelial cells, plated at a density of 1.5×10⁴cells per well in a 24 well plate, were incubated with B19 parvoviruscapsids in the presence of 0.5% fetal calf serum+10.0 ng/ml basicfibroblast growth factor. The various B19 parvovirus capsid preparations(i.e., VP1/2 or capsids made with only VP1 or VP2) were added to eachwell on the following day and the cells with capsids were incubated foradditional 72 hours. Cell proliferation was then determined by using acrystal violet dye assay. Accordingly, capsid treated cells were washedin PBS, fixed in 3.7% formaldehyde, and incubated with crystal violet.The dye was then removed by extensive washes with distilled water. Thecell-associated crystal violet was solubilized with 10% acetic acid andquantified at absorbance 540 nm in an ELISA plate reader.

[0083] FIGS. 11-14 show the results of several endothelial cellproliferation assays, wherein the “x axis” displays an increasingconcentration of control antigen KYVTGIN (SEQ. ID. NO. 1) (FIG. 11), B19parvovirus capsid (VP1 alone) (FIG. 12), B19 parvovirus capsid (VP1/2)FIG. 13), and B19 parvovirus capsid (VP2 alone) (FIG. 14). Thus, fromleft to right, the bars represent the absorbance at 540 nm with 0 μg/ml,0.01 μg/ml, 0.1 μg/ml, 1.0μg/ml, and 10.0 μg/ml. The “y axis” shows astandard of absorbance values at 540 nm. The standard deviation waswithin 10%. As shown in FIG. 14, VP2 capsids efficiently inhibitendothelial cell proliferation at concentrations as low as 1.0 μg/ml andsignificant inhibition was observed at 10. μg/ml.

[0084] The effect of B19 parvovirus capsid preparations on cellmigration was also determined. The migration assays were performed usinga modified Boyden chamber assay (Neuroprobe, Inc.). Basic fibroblastgrowth factor (40 ng/ml) was added to stimulate migration of the HUVECcells through a collagen 1 coated 8 μm pore size millipore filter. Cellswere incubated for 60 min with the various B19 parvovirus capsidpreparations (i.e., VP1/2 or capsids made with only VP1 or VP2) prior toconducting the migration assay. To perform the migration assay, theBoyden chamber was incubated for 4.5 h at 37° C. in a 10% CO₂atmosphere. The filters were subsequently removed and were fixed in 3.7%formaldehyde. Cell migration was visualized by staining the filtersovernight in Gill's Hematoxylin. The number of migrating cells werescored by counting stained cells on the migrating side of the filter perhigh power magnification field. (See FIG. 15). As shown in FIG. 15, VP2capsids at concentrations as low as 1 μg/ml effectively inhibitedendothelial cell migration. Further, the VP2 capsid-mediated inhibitionof endothelial cell migration was significantly more potent than thatobserved with either native capsids (VP1/2) or capsids having only VP1.

[0085] It should be understood that the optimal candidate molecules forthe inhibition of hematopoiesis and cell growth or migration may not bemolecules that completely or even significantly inhibit hematopoiesisand/or cell growth or migration because complete or significantinhibition may have unwanted side effects for a patient, for example. Inmany cases, molecules that only reduce or compromise hematopoiesis, cellproliferation, or migration may be desired. Accordingly, moleculesidentified herein as having any capacity to reduce or inhibit cellgrowth or migration are of significant therapeutic value. As will bediscussed in greater detail below, techniques in protein engineering,computer modeling, epitope mapping, and the “capsid agentcharacterization assays” described herein, can be employed to rapidlymanufacture and identify peptides of varying capacity to inhibithematopoiesis, cell growth, and cell migration.

[0086] The term “capsid agent characterization assays” is intended tomean assays that analyze the ability of a “capsid agent” to inhibit thegrowth of a cell that has the P antigen. Examples of capsid agentsinclude, but are not limited to, a B19 parvovirus VLP or a VP1/2, or VP1or VP2 capsid or modified or unmodified peptide fragments of VP1 or VP2or both or synthetic molecules having sequences of VP1 or VP2 or both orpeptidomimetics that resemble VP1 or VP2 or regions of either or both ofthese molecules. Examples of capsid agent characterization assaysinclude, but are not limited to, colony formation assays, neutralizationassays, protein binding or fusion assays, internalization assays,transcription or translation assays, and assays that evaluate thephosphorylation of proteins or calcium mobilization in a cell aftercontact with a capsid agent. (See also U.S. Pat. No. 5,508,186 to Younget aL, which describes several capsid agent characterization assays).The section below teaches the manufacture and characterization of morecapsid agents that inhibit cell growth and cell migration.

[0087] B19 parvovirus capsid agents that inhibit growth and migration ofcells that have a receptor that interacts with a parvovirus B19 capsidor fragment thereof (e.g., a P antigen containing cell)

[0088] This section describes several techniques that can be used tomanufacture, design, and characterize capsid agents, including but notlimited to, B19 parvovirus capsids, modified B19 parvovirus capsids, B19parvovirus VP2 capsids, fragments of these molecules, andpeptidomimetics that resemble these structures. The VP1 and VP2structural gene has been sequenced in its entirety and this sequence canbe obtained from the NCBI database source accession number U38506.1, oraccession number AAB47788, or medline number 97081188, or as publishedby Erdman et al., J. Gen. Virol., 77: 2767 (1996), all references andsequences therein are hereby expressly incorporated by reference. TheVP1 or VP2 or fragments of either or both used with embodiments of theinvention correspond to sequences involved in the inhibition of cellgrowth and cell migration. Desirable peptides consist, consistessentially of, or comprise between three amino acids and 780 aminoacids of the VP1 and VP2 structural protein but have at least someportion of the molecule that is involved in the inhibition of growthand/or migration of cells that have a receptor that interacts with aparvovirus B19 capsid or fragment thereof (e.g., a P antigen containingcell). In other words, preferable embodiments of the invention concernat least three amino acids, four amino acids, five amino acids, sixamino acids, seven amino acids, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen,twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five,twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one,thirty-two, thirty-three, thirty-four, thirty-five, thirty-six,thirty-seven, thirty-eight, thirty nine, or forty or fifty or sixty orseventy or eighty or ninety or one-hundred amino acids of either the VP1or the VP2 structural gene or both. Desirable embodiments concern atleast 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510,520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650,660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, or 780 aminoacids of the VP1 or VP2 structural protein or both.

[0089] The peptides and fragments or derivatives thereof that areinvolved in the inhibition of growth and migration of cells that havethe P antigen, include but are not limited to, those regions of the VP1and VP2 structural gene that is found in nature. Additionally, alteredsequences in which functionally equivalent amino acid residues aresubstituted for residues within the sequence resulting in a silentchange can also be present in these capsid agents. In some aspects ofthe invention, the term “consisting essentially of” encompasses themolecules described above because the changes made to the capid agentsare not material alterations. That is, one or more amino acid residueswithin the sequence of the VP1 or VP2 structural gene, or a fragmentthereof can be substituted by another amino acid of a similar polaritythat acts as a functional equivalent, resulting in a silent alteration.Substitutes for an amino acid within the sequence can be selected fromother members of the class to which the amino acid belongs. For example,the non-polar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.The uncharged polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine and glutamine. The positivelycharged (basic) amino acids include arginine, lysine, and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. The aromatic aminoacids include phenylalanine,tryptophan, and tyrosine. The peptides described above are preferablyanalyzed in assays to determine whether the fragment has retained theability to inhibit the growth and/or migration of cells that have areceptor that interacts with a parvovirus B19 capsid or fragment thereof(e.g., a P antigen containing cell).

[0090] Peptides for use in aspects of the invention can also bemodified, e.g., the peptides can have substituents not normally found ona peptide or the peptides can have substituents that are normally foundon the peptide but are incorporated at regions of the peptide that arenot normal. These peptides can be acetylated, acylated, or aminated, forexample. Substituents that can be included on the peptide so as tomodify it include, but are not limited to, H, alkyl, aryl, alkenyl,alkynl, aromatic, ether, ester, unsubstituted or substituted amine,amide, halogen or unsubstituted or substituted sulfonyl or a 5 or 6member aliphatic or aromatic ring. The fragments described herein, forexample, can have a carboxy terminal amide or can have one or more Damino acids or can be retroinverso peptides. In some embodiments, theterm “consisting essentially of” encompasses the modified capsid agentsabove.

[0091] Additionally, VP1 or VP2 or fragments of either or both can bederivatized in that the derivative polypeptide can be manipulated toinclude amino acid sequences that effect the function and stability ofthe molecule. For example, peptides that are involved in the inhibitionof growth and migration of cells that have the P antigen can beengineered to have one or more cysteine residues so as to promote theformation of a more stable derivative through disulfide bond formation.(See e.g., U.S. Pat. No. 4,908,773). Computer graphics programs and theassays described herein can be employed to identify cystine linkagesites that provide greater stability but do not perturb the ability toinhibit growth or migration of cells that have a receptor that interactswith a parvovirus B19 capsid or fragment thereof (e.g., a P antigencontaining cell). (See e.g., Perry, L. J., & Wetzel, R., Science,226:555-557 (1984); Pabo, C. O., et al., Biochemistry, 25:5987-5991(1986); Bott, R., et al., European Patent Application Ser. No. 130,756;Perry, L. J., & Wetzel, R., Biochemistry, 25:733-739 (1986); Wetzel, R.B., European Patent Application Ser. No. 155,832).

[0092] Additional derivatives that are embodiments of the inventioninclude peptidomimetics that resemble regions of VP1, VP2, or both.Synthetic peptides can be prepared that correspond to these molecules byemploying conventional synthetic methods, utilizing L-amino acids,D-amino acids, or various combinations of amino acids of the twodifferent configurations. Synthetic compounds that mimic theconformation and desirable features of a particular peptide but avoidthe undesirable features, e.g., flexibility (loss of conformation) andbond breakdown are known as “peptidomimetics”. (See, e.g., Spatola, A.F. Chemistry and Biochemistry of Amino Acids. Peptides, and Proteins(Weistein, B, Ed.), Vol. 7, pp. 267-357, Marcel Dekker, New York (1983),which describes the use of the methylenethio bioisostere [CH₂ S] as anamide replacement in enkephalin analogues; and Szelke et al., Inpeptides: Structure and Function, Proceedings of the Eighth AmericanPeptide Symposium, (Hruby and Rich, Eds.); pp. 579-582, Pierce ChemicalCo., Rockford, Ill. (1983), which describes renin inhibitors having boththe methyleneamino [CH₂ NH] and hydroxyethylene [CHOHCH₂ ] bioisosteresat the Leu-Val amide bond in the 6-13 octapeptide derived fromangiotensinogen). Numerous methods and techniques are known in the artfor designing and manufacturing peptidomimetcs, any of which could beused. (See, e.g., Farmer, P. S., Drug Design, (Ariens, E. J. ed.), Vol.10, pp. 119-143 (Academic Press, New York, London, Toronto, Sydney andSan Francisco) (1980); Farmer, et al., in TIPS, 9/82, pp. 362-365;Verber et al., in TINS, 9/85, pp. 392-396; Kaltenbronn et al., in J.Med. Chem. 33: 838-845 (1990); and Spatola, A. F., in Chemistry andBiochemistry of Amino Acids. Peptides, and Proteins, Vol. 7, pp.267-357, Chapter 5, “Peptide Backbone Modifications: AStructure-Activity Analysis of Peptides Containing Amide BondSurrogates. Conformational Constraints, and Relations” (B. Weisten, ed.;Marcell Dekker: New York, pub.) (1983); Kemp, D. S., “Peptidomimeticsand the Template Approach to Nucleation of beta.-sheets andalpha.-helices in Peptides,” Tibech, Vol. 8, pp. 249-255 (1990).Additional teachings can be found in U.S. Pat. Nos. 5,288,707;5,552,534; 5,811,515; 5,817,626; 5,817,879; 5,821,231; and 5,874,529,herein incorporated by reference. Accordingly, peptidomimetics of theinvention can have structures that resemble between at least 3 and 780amino acids. That is, they can resemble 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320,330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460,470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600,610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740,750, 760, 770, or 780 amino acids of the VP1 or VP2 structural proteinso long as some region of the molecule inhibits the growth or migrationof a cell that has a receptor that interacts with a parvovirus B19capsid or fragment thereof (e.g., a P antigen containing cell).

[0093] Conventional techniques in molecular biology, such as thosedescribed in U.S. Pat. No. 5,508,186, herein expressly incorporated byreference in its entirety, can be used to prepare numerous types ofcapsid agents. The term “capsid agents” can refer to capsids comprisingVP1, VP2, VP1/2 in varying proportions, fragments of VP1 or VP2 oreither or both, fusion proteins having sequences that correspond to VP1or VP2 or both, and modified or unmodified proteins or peptides orpeptidomimetics that correspond to sequences of the VP1 and VP2structural gene that are involved in the inhibition of growth and/ormigration of cells that have a receptor that interacts with a parvovirusB19 capsid or fragment thereof (e.g., a P antigen containing cell).

[0094] By the approach described in U.S. Pat. No. 5,508,186 a capsidagent can be manufactured as follows. Plasmids can be constructed tocontain either full length VP1 or VP2 or both. To construct plasmidpVP1/941, a CDNA encoding the VP1 gene can be excised from pYT103c, anearly full length molecular clone of B19 parvovirus (Cotmore et al.Science 226:1161 (1984); Ozawa et al. J. Virol. 62:2884 )1988)), bydigestion with the restriction enzymes Hind III (which cuts at map unit45) and EcoRI (which cuts at map unit 95) followed by treatment withmung bean nuclease to complement single stranded ends. The resultant DNAfragment is then inserted into the BamHI site (made blunt ended with theKlenow fragment of DNA polymerase) of the baculovirus transfer vectorpVL941, a vector derived by deletion of the polyhedrin gene of AcMNPV(Autographa california nuclear polyhedrosis virus) followed by cloninginto the pUC8 plasmid (Summers et al. Tex. Agric. Exp. Stn. 1555(1987)). Construction of pVP2/941 is performed by the insertion of aPstI-EcoRI digestion fragment of pYT103c (map units 58-95; the EcoRIsite was blunt-ended) and a synthetic DNA fragment of 20 nucleotidescorresponding to the SstI-PstI region (again with the SstI siteblunt-ended) into the BamHI site of pVL941. Additionally, the PolymeraseChain Reaction (PCR) can be used to clone the VP1 or VP2 gene orportions thereof from full-length clones as described by Erdman et al.,J. Gen Virol. 77:2767 (1996), herein incorporated by reference in itsentirety. To facilitate cloning, the primers can be designed to generateconvenient sites for restriction digestion, as is known in the art.

[0095] Recombinant plasmids encoding VP1, VP2, VP1/2, or fragmentsthereof are then transfected into insect cells to generate recombinantbaculoviruses. Accordingly, 8 μg of the recombinant plasmid iscotransfected into Sf9 cells with 2 μg of wild type AcMNPV, usingcalcium phosphate-mediated precipitation. The Sf9 cell line (AmericanType Culture Collection, Rockville Md.), which is derived fromSpodoptera frugiperda (fall army worm) ovary, is maintained in Grace'sinsect tissue culture medium containing 10% heat inactivated fetalbovine serum, 2.5 μg/ml fungizone, 50 μ/ml gentamicin, 3.33 mg/mllactalbumin hydrolysate, and 3.33 mg/ml yeastolate (provided complete byGibco BRL Life Technologies, Gaithersburg Md.) at 100% room air, 95%humidity, at 27° C. Six days after transfection, progeny virus isharvested and replaqued onto fresh Sf9 cells. Recombinant viruses arerecognized visually by the absence of occlusion bodies in the nucleus ofcells (the occlusion-positive phenotype is the result of synthesis oflarge quantities of the polyhedrin protein). Recombinant viruses can besubjected to three cycles of plaque purification before large scale VLPstocks are prepared and isolated or purified. Purified compositionscontaining 0.1%, 0.5%, 1%, 2%, 5%, 10%, 25%, or more (weight/weight) ofthe active ingredient are specifically contemplated.

[0096] The term “isolated” requires that the material be removed fromits original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally occurring protein presentin a living cell is not isolated, but the same protein, separated fromsome or all of the coexisting materials in the natural system, isisolated. The term “purified” does not require absolute purity; ratherit is intended as a relative definition. For example, proteins areroutinely purified to electrophoretic homogeneity, as detected byCoomassie staining, and are suitable in several assays despite havingthe presence of contaminants. Preferably, capsid agent characterizationassays are performed on the isolated or purified capsid agentsincluding, but not limited to, the assays described in U.S. Pat. No.5,508, 186 (e.g., DNA, RNA, and proten analysis, immunoblots,immunofluorescence, sedimentation analysis, electron microscopy, immuneelectron microscopy, and the capsid agent characterization assaysdescribed previously.

[0097] In some embodiments, particularly for applications that involvethe long-term administration of capsid agents, it is desirable tomanufacture a pharmaceutical that does not elicit a significant immuneresponse in a subject. A general scheme for the manufacture of capsidagents that do not induce an immune response involves design of theagent, construction of the agent, analysis of the agent's ability toinhibit cell growth and/or cell migration and an analysis of the immuneresponse generated to the agent. Many of the immunogenic regions of theB19 parvovirus capsid are known and, through conventional techniques inmolecular biology, these immunogenic regions can be deleted,mutagenized, or modified and the newly designed synthetic capsidproteins can be analyzed in one or more capsid agent characterizationassays (e.g., a colony formation assay and a neutralization assay usingsera generated from asymptomatic individuals). Many methods can beemployed to identify the immunogenic regions of the B19 parvoviruscapsid and manufacture non-immunogenic VLPs that inhibit cell growthand/or migration and the example below is provided as one possibleapproach.

[0098] Test expression constructs can be designed, manufactured, andanalyzed as follows. This process can be iterative so as to generateseveral classes of VLPs and pharmaceuticals having these capsid agents,which differ according to their ability to inhibit cell growth, cellmigration, and induce an immune response in a subject. Accordingly, byone approach, the VP2 structural gene can be cloned from clinicalisolates using PCR with primers designed from the published VP2sequence. The VP2 gene is subsequently subcloned both into BlueScript(Pharmacia) for mutagenesis, and pVL1393 (Stratagene) for expression inSf9 cells. Mutations that correspond to immunogenic regions of VP2(e.g., amino acids 253-272, 309-330, 328-344, 359-382, 449-468, and491-515) are introduced into the VP2 gene using Amersham Sculptor invitro mutagenesis kit. One of skill in the art will appreciate thatcarboxy truncations, amino truncations, internal truncations, andsite-directed mutagenesis of the VP1 and VP2 structural protein can beaccomplished by several approaches. Preferably, several different cloneshaving one or more of the deletions described above are generated. Theappearance of a desired mutation is confirmed by sequencing and themutated gene is then subcloned into pVL1393 for expression in Sf9 cells.The SF9 cells are then transfected using BaculoGold Transfection kit(Pharmingen). Transfections can be performed according to themanufacturer's instructions with the following modifications.Approximately, 8×10⁸ Sf9 cells are transfected in a 100 mM dish, with 4μg of BaculoGold DNA and 6 μg of test DNA. Cells are harvested after 6days and assayed for VLP production.

[0099] Next, cells are harvested by scraping followed by low speedcentrifugation. Cells are then resuspended in 300 ml of breaking buffer(1 M NaCl, 0.2 M Tris pH 7.6) and homogenized for 30 seconds on iceusing a Polytron PT 1200 B with a PT-DA 1205/2-A probe (Brinkman) in aFalcon 1259 tube. Samples are spun at 2500 rpm for 3 minutes to pelletdebris and the tubes are washed with an additional 150 ml of breakingbuffer. The supernatants are collected in a 1.5 ml microfuge tubes andare re-spun for 5 minutes in an Eppendorf microfuge (Brinkman). Thecollected supernatants can be stored at 4° C.

[0100] ELISA assays can then be performed on the isolated VLPs asfollows. Approximately, 5 ml of extract is diluted into 50 ml of 1% BSAin PBS (phosphate buffered saline; 20 mM NaPO₄, pH 7.0, 150 mM NaCl) andis plated onto a polystyrene plate. The plate is incubated overnight at4° C. Extracts are removed and the plate is blocked with 5% powderedmilk in PBS. All subsequent wash steps are performed with 1% BSA in PBS.The plate is incubated at room temperature with primary antibody for 1hour (e.g., sera generated from asymptomatic individuals). After washingto remove unbound antibody, plates are incubated for 1 hour withsecondary antibody. The secondary antibody, peroxidase labeled Goatanti-Mouse IgG (g), can be purchased from Kirkegaard & PerryLaboratories, Inc. and can be used at 10³ dilution in 1% BSA in PBS.After a final washing, an alkaline phosphatase assay is performed andabsorbance is read at 405 nm. The most successful capsid agents by thisassay will be ones that evade detection. That is, desired mutant VP2capsids are ones that have lost epitopes recognized by antibodiespresent in the sera and, thus, are not detected by the ELISA. Byperforming these experiments with several lots of sera obtained fromdifferent individuals and the monoclonal antibodies that neutralize theinhibition of colony formation or cell migration, one of skill canrapidly identify the regions of VP2 that are immunogenic and mutant VP2capsids that best evade an immune response.

[0101] Next, the mutant VP2 capsids that successfully evade detection bythe ELISA method described above are analyzed for their ability toinhibit cell growth and cell migration by using a capsid agentcharacterization assays. By assessing each mutant VP2 capsid's abilityto inhibit cell growth and cell migration and coordinating thisinformation with the immunogenicity results from the ELISA analysis, “acapsid agent profile” can be generated. A “capsid agent profile” caninclude a symbol or icon that represents a mutant capsid protein ormutant VLP, sequence information (e.g., the location of mutations ormodifications), a capsid agent class designation (e.g., informationregarding relationships to other capsid agents), application information(e.g., disease indications or treatment information, or clinical orbiotechnological uses), and performance information from capsid agentcharacterization assays (e.g., values obtained from the colony formationassays, neutralization assays, fusion/intemalization assays, bindingassays, phosphorylation assays, cell migration assays, proliferationassays, and results obtained from immunogenicity analysis including theELISA assays).

[0102] Capsid agent profiles can be recorded on a computer readablemedia, stored in a database, on hardware, software, or memory, accessedwith a search engine and can be compared with one another or associatedwith a disease state or “disease state profile”, which is informationrelating to a disease, condition or indicated treatment. These capsidagent profiles and disease state profiles can be used by investigatorsfor rational drug design or biochemical analysis or by physicians orclinicians who wish to choose an appropriate pharmaceutical compositionthat balances the optimal level of cell growth and cell migrationinhibition with immune response of the subject in light of the desiredduration of treatment.

[0103] In several embodiments, the capsid agents are disposed on asupport so as to create a multimeric capsid agent. While a monomericagent (that is, an agent that presents a discrete molecule, thus,carrying only one binding domain) can be sufficient to achieve a desiredresponse, a multimeric agent (that is, an agent that presents multiplemolecules, thus, having several domains) often times can elicit agreater response. It should be noted that the term “multimeric” refersto the presence of more than one molecule on a support, for example,several individual molecules of B19 parvovirus VP2 capsid joined to asupport, as distinguished from the term“multimerized” that refers to anagent that has more than one molecule joined as a single discretecompound molecule on a support, for example several molecules of B19parvovirus VP2 capsid joined to form a single compound molecule that isjoined to a support. A multimeric form of the capsid agents describedherein can be advantageous for many biotechnological or clinicalapplications because of the ability to obtain an agent with higheraffinity for a cell having a receptor that interacts with a parvovirusB19 capsid or fragment thereof (e.g., a P antigen containing cell).

[0104] A multimeric capsid agent can be obtained by coupling theprotein, for example, B19 parvovirus VP2 capsid or a fragment thereof toa macromolecular support. A “support” may also be termed a carrier, aresin or any macromolecular structure used to attach or immobilize aprotein. The macromolecular support can have a hydrophobic surface thatinteracts with regions of the capsid agent by hydrophobic non-covalentinteractions. The hydrophobic surface of the support can be, forexample, a polymer such as plastic or any other polymer in whichhydrophobic groups have been linked such as polystyrene, polyethylene,PTFE, or polyvinyl. Alternatively, capsid agents can be covalently boundto carriers including proteins and oligo/polysaccarides (e.g. cellulose,starch, glycogen, chitosane or aminated sepharose). In these laterembodiments, a reactive group on capsid agent, such as a hydroxy or theamino present in the peptide, can be used to join to a reactive group onthe carrier so as to create the covalent bond. Embodiments also cancomprise a support with a charged surface that interacts with the capsidagent. Additional embodiments concern a support that has other reactivegroups that are chemically activated so as to attach a capsid agent. Forexample, cyanogen bromide activated matrices, epoxy activated matrices,thio and thiopropyl gels, nitrophenyl chloroformate and N-hydroxysuccinimide chlorformate linkages, or oxirane acrylic supports can beused. (SIGMA).

[0105] Further, the support can comprise inorganic carriers such assilicon oxide material (e.g. silica gel, zeolite, diatomaceous earth oraminated glass) to which the capsid agent is covalently linked through ahydroxy, carboxy or amino group of the peptide and a reactive group onthe carrier. Thus, in appropriate contexts, a “support” can refer to thewalls or wells of a reaction tray, test tubes, catheters, stents,balloons, prosthetics, medical devices, polystyrene beads, magneticbeads, nitrocellulose strips, membranes, microparticles such as latexparticles, sheep (or other animal) red blood cells, Duracyte® artificialcells, and others. Inorganic carriers, such as silicon oxide material(e.g. silica gel, zeolite, diatomaceous earth or aminated glass) towhich the capsid agents are covalently linked through a hydroxy, carboxyor amino group and a reactive group on the carrier are also embodiments.Carriers for use in the body, (e.g., for prophylactic or therapeuticapplications) are preferably physiological, non-toxic andnon-immunoresponsive. Such carriers include, but are not limited to,poly-L-lysine, poly-D, L-alanine and Chromosorb® (Johns-ManvilleProducts, Denver Colo.).

[0106] In other embodiments, linkers, such as λ linkers or biotin-avidin(or streptavidin), of an appropriate length are inserted between thecapsid agent and the support so as to encourage greater flexibility andthereby overcome any steric hindrance that is presented by the support.The determination of an appropriate length of linker that allows foroptimal interaction is made by screening the capsid agents havingvarying length linkers in the capsid agent characterization assaysdescribed herein.

[0107] In other embodiments, the multimeric supports discussed abovehave attached multimerized capsid agents so as to createa“multimerized-multimeric support”. An embodiment of a multimerizedcapsid agent is obtained by creating an expression construct having twoor more nucleotide sequences encoding VP2 or a fragment thereof, forexample, joined together. The expressed fusion protein is one embodimentof a multimerized capsid agent and is then joined to a support. Asupport having many such multimerized agents is termed amultimerized-multimeric support. Linkers or spacers between the domainsthat make-up the multimerized agent and the support can be incorporatedfor some embodiments and optimally spaced linkers can be determinedusing the capsid agent characterization assays.

[0108] In some embodiments, capsid agents are disposed on prostheticdevices that are implanted into a subject. With many types ofprosthetics, for example, stents and valves, a limited amount of tissueingrowth is desired so as to stabilize the implant. During implantation,however, the injury to surrounding tissue results in a considerableincrease in cellular proliferation, which can cause fibrotic build up orrestenosis and, over time, constriction of a stent or repositioning of avalve. Prior art devices have sought to overcome this problem throughthe use of radioactivity, however, the treatment success and potentialfor systemic exposure to the radioactive substances that are releasedfrom the device makes such approaches less than desirable. Similarly,oftentimes techniques such as balloon angioplasty result in restenosiscaused by the infiltration of endothelial cells.

[0109] By attaching capsid agents to medical prosthetics, such as stentsor valves, or delivering capsid agents through porous catheters (e.g.,balloon cathers as used in angioplasty) endothelial cell migration,proliferation, fibrotic build up, tissue ingrowth, and restenosis can beefficiently inhibited. Further, a delayed tissue ingrowth can beobtained by using capsid agents that are cleared by the immune system ata time after the inflammation associated with the medical procedure hasquelled. By using the approaches described above, capsid agents can beattached to many different types of prosthetics, e.g., stents or valves,through hydrophobic interactions or covalent linkages. Further, cathersin the prior art can be adapted for the delivery of capsid agents to thesite of angioplasty. By analyzing the capsid agent profiles, a physiciancan select the appropriate capsid agent-coated prosthetic forimplantation or the appropriate capsid agent for delivery depending onthe desired time of cell inhibition or delay in tissue ingrowth.Localized delivery of capsid agents in other manners is alsocontemplated. Thus, for example, growth of vascular endothelial cellscan be inhibited by implanting a controlled release composition in thevicinity of a stent, graft, valve, or other prosthetic, or by deliveringthe drug to the site via infusion pump or other suitable device. Inaddition to coatings for medical devices and formulations for catheterdelivery, the capsid agents described herein can be formulated inpharmaceuticals and used to treat or prevent human diseases orconditions associated with proliferation or migration of cells that havethe P antigen. The section below discusses the many ways to formulatecapsid agents into pharmaceuticals and determine an appropriate dose.

[0110] The manufacture and dose of therapeutic and prophylactic agents

[0111] The capsid agents of the invention (e.g., B19 parvovirus VP1,VP1/2, VP2 capsids or fragments thereof) are suitable for treatment ofsubjects either as a preventive measure to avoid a disease or condition,or as a therapeutic to treat subjects already afflicted with a disease.These pharmacologically active compounds can be processed in accordancewith conventional methods of galenic pharmacy to produce medicinalagents for administration to subjects, e.g., mammals including humans.The active ingredients can be incorporated into a pharmaceutical productwith and without modification. Further, the manufacture ofpharmaceuticals or therapeutic agents that deliver the pharmacologicallyactive compounds described herein by several routes are aspects of theinvention. For example, and not by way of limitation, DNA, RNA, andviral vectors having sequences encoding the capsid agents are used withembodiments. Nucleic acids encoding capsid agents can be administeredalone or in combination with other active ingredients.

[0112] The compounds of this invention can be employed in admixture withconventional excipients, i.e., pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral, enteral (e.g.,oral) or topical application that do not deleteriously react with thepharmacologically active ingredients of this invention. Suitablepharmaceutically acceptable carriers include, but are not limited to,water, salt solutions, alcohols, gum arabic, vegetable oils, benzylalcohols, polyetylene glycols, gelatine, carbohydrates such as lactose,amylose or starch, magnesium stearate, talc, silicic acid, viscousparaffin, perfume oil, fatty acid monoglycerides and diglycerides,pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinylpyrrolidone, etc. Many more suitable vehicles are described inRemmington's Pharmaceutical Sciences, 15th Edition, Easton:MackPublishing Company, pages 1405-1412 and 1461-1487(1975) and The NationalFormulary XIV, 14th Edition, Washington, American PharmaceuticalAssociation (1975), herein incorporated by reference. The pharmaceuticalpreparations can be sterilized and if desired mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, coloring,flavoring and/or aromatic substances and the like that do notdeleteriously react with the active compounds.

[0113] The effective dose and method of administration of a particularpharmaceutical formulation can vary based on the individual patient andthe type and stage of the disease, as well as other factors known tothose of skill in the art. Therapeutic efficacy and toxicity of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., ED50 (the dosetherapeutically effective in 50% of the population). Any monkey specieswith a receptor which makes them permissive to the B19 capsid effect areappropriate experimental models, as described earlier. The data obtainedfrom cell culture assays and animal studies is used in formulating arange of dosage for human use. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED50 with no toxicity. The dosage varies within this range dependingupon type of capsid agent, the dosage form employed, sensitivity of thepatient, and the route of administration.

[0114] Normal dosage amounts may vary from approximately 1 to 100,000micrograms, up to a total dose of about 10 grams, depending upon theroute of administration. Desirable dosages include 250 μg, 500 μg, 1 mg,50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg,500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg,1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2 g,3 g, 4 g, 5, 6 g, 7 g, 8 g, 9 g, and 10 g. Additionally, theconcentrations of the capsid agents can be quite high in embodimentsthat administer the agents in a topical form. Molar concentrations ofcapsid agents can be used with some embodiments. Desirableconcentrations for topical administration and/or for coating medicalequipment range from 100 μM to 8 mM. Preferable concentrations for theseembodiments range from 500 μM to 500 mM. For example, preferredconcentrations for use in topical applications and/or for coatingmedical equipment include 500 μM, 550 μM, 600 μM, 650 μM, 700 μM, 750μM, 800 μM, 850 μM, 900 μM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 3mM, 35 mM, 40 mM, 45 mM, 50 mM, 60 mM, 7 mM, 80 mM, 90 mM, 100 mM, 120mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 300mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, and 500 mM.

[0115] In some embodiments, the dose of capsid agent preferably producesa tissue or blood concentration or both from approximately 0.1 μM to 500mM. Desirable doses produce a tissue or blood concentration or both ofabout 1 to 800 μM. Preferable doses produce a tissue or bloodconcentration of greater than about 10 μM to about 500 μM. Preferabledoses are, for example, the amount of capsid agent required to achieve atissue or blood concentration or both of 10 μM, 15 μM, 20 μM, 25 μM, 30μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80μM, 85 μM, 90 μM, 95 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 145 μM,150 μM, 160 μM, 170 μM, 180 μM, 190 μM, 200 μM, 220 μM, 240 μM, 250 μM,260 μM, 280 μM, 300 μM, 320 μM, 340 μM, 360 μM, 380 μM, 400 μM, 420 μM,440 μM, 460 μM, 480 μM, and 500 μM. Although doses that produce a tissueconcentration of greater than 800 μM are not preferred, they can be usedwith some embodiments of the invention. A constant infusion of thecapsid agent can also be provided so as to maintain a stableconcentration in the tissues as measured by blood levels.

[0116] The exact dosage is chosen by the individual physician in view ofthe patient to be treated. Dosage and administration are adjusted toprovide sufficient levels of the active moiety or to maintain thedesired effect. Additional factors that can be taken into accountinclude the severity of the disease state of the patient, age, andweight of the patient; diet, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Short acting pharmaceutical compositions can be administereddaily whereas long acting pharmaceutical compositions can beadministered every 2, 3 to 4 days, every week, or once every two weeks.It is also contemplated that short acting compositions could have longterm effects in that the compositions may have an immediate effect onhematopoiesis that takes the body several days or weeks to recover.Depending on half-life, clearance rate of the particular formulation,and the time takes an individual to recover from the inhibition ofhematopoiesis, for example, the pharmaceutical compositions of theinvention can be administered once, twice, three, four, five, six,seven, eight, nine, ten or more times per day, week, or month.

[0117] Routes of administration of the pharmaceuticals of the inventioninclude, but are not limited to, transdermal, parenteral,gastrointestinal, transbronchial, and transalveolar. Transdermaladministration is accomplished by application of a cream, rinse, gel,etc. capable of allowing the pharmacologically active compounds topenetrate the skin. Parenteral routes of administration include, but arenot limited to, electrical or direct injection such as direct injectioninto a central venous line, intravenous, intramuscular, intraperitoneal,intradermal, or subcutaneous injection. Gastrointestinal routes ofadministration include, but are not limited to, ingestion and rectal.Transbronchial and transalveolar routes of administration include, butare not limited to, inhalation, either via the mouth or intranasally.

[0118] Compositions having the pharmacologically active compounds ofthis invention that are suitable for transdermal administration include,but are not limited to, pharmaceutically acceptable suspensions, oils,creams, and ointments applied directly to the skin or incorporated intoa protective carrier such as a transdermal device (“transdermal patch”).Examples of suitable creams, ointments, etc. can be found, for instance,in the Physician's Desk Reference. Examples of suitable transdermaldevices are described, for instance, in U.S. Pat. No. 4,818,540 issuedApr. 4, 1989 to Chinen, et al., herein incorporated by reference.

[0119] Compositions having the pharmacologically active compounds ofthis invention that are suitable for parenteral administration include,but are not limited to, pharmaceutically acceptable sterile isotonicsolutions. Such solutions include, but are not limited to, saline andphosphate buffered saline for injection into a central venous line,intravenous, intramuscular, intraperitoneal, intradermal, orsubcutaneous injection.

[0120] Compositions having the pharmacologically active compounds ofthis invention that are suitable for transbronchial and transalveolaradministration include, but not limited to, various types of aerosolsfor inhalation. Devices suitable for transbronchial and transalveolaradministration of these are also embodiments. Such devices include, butare not limited to, atomizers and vaporizers. Many forms of currentlyavailable atomizers and vaporizers can be readily adapted to delivercompositions having the pharmacologically active compounds of theinvention.

[0121] Compositions having the pharmacologically active compounds ofthis invention that are suitable for gastrointestinal administrationinclude, but not limited to, pharmaceutically acceptable powders, pillsor liquids for ingestion and suppositories for rectal administration.Due to the ease of use, gastrointestinal administration, particularlyoral, is a preferred embodiment. Once the pharmaceutical comprising thecapsid agent has been obtained, it can be administered to a subject inneed to treat or prevent diseases or conditions associated withproliferation or migration of a cell that has a receptor that interactswith a parvovirus B19 capsid or fragment thereof (e.g., a P antigencontaining cell).

[0122] Aspects of the invention also include a coating for medicalequipment such as prosthetics, implants, and instruments. Coatingssuitable for use in medical devices can be provided by a gel or powdercontaining the capsid agents or by polymeric coating into which thecapsid agents are suspended. Suitable polymeric materials for coatingsor devices are those that are physiologically acceptable and throughwhich a therapeutically effective amount of the capsid agent candiffuse. Suitable polymers include, but are not limited to,polyurethane, polymethacrylate, polyamide, polyester, polyethylene,polypropylene, polystyrene, polytetrafluoroethylene, polyvinyl-chloride,cellulose acetate, silicone elastomers, collagen, silk, etc. Suchcoatings are described, for instance, in U.S. Pat. No. 4,612,337, issuedSep. 16, 1986 to Fox et al. that is incorporated herein by reference inits entirety. The section below describes several methods to treatdiseases or conditions associated with proliferation or migration of acell that has a receptor that interacts with a parvovirus B19 capsid orfragment thereof (e.g., a P antigen containing cell), using apharmaceutical having a capsid agent as an active ingredient.

[0123] Therapeutic and prophylactic approaches

[0124] In several aspects of the invention, capsid agents, in particularpharmaceuticals having capsid agents, are provided to a subject in needto treat or prevent a disease or condition associated with abnormal cellproliferation and/or cell migration. Methods to formulatepharmaceuticals for the inhibition of growth or migration of cells thathave a receptor that interacts with a parvovirus B19 capsid or fragmentthereof (e.g., a P antigen containing cell), including, but not limitedto, hematopoietic cells and endothelial cells, are embodiments of theinvention. That is, some embodiments include the use of medicamentscomprising a capsid agent for the inhibition of growth and/or migrationof cells that have a receptor that interacts with a parvovirus B19capsid or fragment thereof (e.g., a P antigen containing cell), such ashematopoeitic cells and endothelial cells.

[0125] In one embodiment, for example, capsid agents can be used toinhibit hematopoesis in recipient subjects prior to in utero stem celltransplantation. In a previous study on tissue distribution of stemcells in the human fetus, it was estimated that a fetal transplantationwith 5×10⁷ cells in the second trimester would produce adonor-to-recipient ratio of approximately 1:1000-1:10000. Such a lowratio fails to provide transplanted cells with a competitive edge overthe native stem cells. (Westgren et al., Am J Obstet Gynecol, 176:49(1996)). To improve this ratio and the success of stem celltransplantation, capsid agents can be administered prior totransplantation so as to suppress the native stem cell population andthereby improve the transplantation. Furthermore, treatment of donorstem cells with anti-P monoclonal antibodies prior to transplantationcan protect them from suppression by the capsid agents, and therebyprovide an even more favorable status. Thus, one embodiment includes amedicament comprising a capsid agent for treatment of a patient prior tostem cell transplantation. This method of treatment can be performed byidentifying a subject in need of an in utero stem cell transplantationand providing to said subject a therapeutically beneficial amount of acapsid agent that inhibits hematopoietic cell growth.

[0126] In a similar aspect of the invention, a method ofnon-myeloablative conditioning prior to postnatal stem celltransplantation is embodied. Recently, methods of nonmyeloblativeconditioning have received considerable attention because such protocolsare less toxic to the patients than the standard approach, whichinvolves high-dose chemo-radiotherapy. (Giralt et al., Blood, 89:4531(1997); Slavin et al., Blood, 91:756 (1998)). However, complete donorhematopoietic chimerism using existing techniques in non-myeloablativetherapy has not been very successful. By providing capsid agents priorto postnatal stem cell transplantation, the ratio of donor cells torecipient cells can be favorably skewed and donor hematopoieticchimerism can be achieved without radiation. Accordingly, a method ofnon-myeloablative conditioning can be performed by identifying a subjectin need of non-myeloablative conditioning prior to postnatal stem celltransplantation and administering to said subject a therapeuticallybeneficial amount of a capsid agent.

[0127] Still another aspect of the invention is directed to a method oftreating a subject suffering from an hematological proliferativedisorders, e.g., Polycythemia Vera. Polycythemia Vera (PCV) is ahaematological disease caused by an uncontrolled proliferation of redblood cells in the bone marrow. Cells of other lineage (leukocytes andthrombocytes) are involved in some patients and may also give rise tosevere complications. The disease is normally seen in middle-aged andaged individuals (median age at diagnosis is 60 years) and the incidencein Sweden is 1.5 cases per 100,000 inhabitants. To date, there is nospecific pharmacological treatment and current approaches to the problemseek to ease the symptoms of the slowly progressing disease. Mediansurvival time without treatment is short. In younger individuals, withoptimal treatment, one can obtain a reasonable quality of life forperiods up to 20 years.

[0128] By administering capsid agents to subjects suffering from PCV,the proliferation of hematopoietic cells can be inhibited and aneffective treatment for this deadly disease can be provided.Accordingly, a method of PCV can be performed by identifying a subjectin need of treatment for PCV and administering to said subject atherapeutically beneficial amount of a capsid agent. Because a long-termtreatment protocol is envisioned, preferably, the capsid agents used areones that elicit a minimal immune response.

[0129] Yet another aspect of the invention is directed to a method oftreating a patient for inhibition of endothelial cell growth. Asdescribed above, undesired endothelial cell growth can occur aftersurgical trauma, e.g., after the implantation of a valve, stent or otherprosthetic or angioplasty, in said patient. Additionally, tumordevelopment and metastasis requires endothelial cell growth and cellmigration. Thus, embodiments of the invention concern medicaments thatinhibit cancer, more specifically, angiogenesis and the cell migrationevents associated with metastasis.

[0130] Angiogenesis concerns the formation of new capillary bloodvessels by a process of sprouting from pre-existing vessels.Angiogenesis occurs during development, as well as in a number ofphysiological and pathological settings, and is necessary for tissuegrowth, wound healing, female reproductive function, and is a componentof pathological processes such as hemangioma formation and ocularneovascularization. However, much of the longstanding interest inangiogenesis comes from the discovery that solid tumors must undergoangiogenesis inorder to grow beyond a critical size. That is, tumorsmust recruit endothelial cells from the surrounding stroma to form theirown endogenous microcirculation.

[0131] By administering capsid agents to subjects suffering from cancer,the proliferation and migration of endothelial cells can be inhibitedand, thus, tumorigenesis and metastasis can be prevented. Accordingly, amethod of inhibiting angiogenesis, tumorigenesis, or cancer can beperformed by identifying a subject in need of an inhibition inangiogenesis, tumorigenesis, or cancer and administering to said subjecta therapeutically beneficial amount of a capsid agent. Because along-term treatment protocol is envisioned, preferably, the capsidagents used are ones that elicit a minimal immune response.

[0132] Additional embodiments of the invention include kits containingcapsid agents, and written instructions for dosage and administration toa patient for hematopoietic progenitor cell growth inhibition,instructions for dosage and administration for hematopoietic progenitorcell growth inhibition in a patient prior to stem cell transplantationto said patient, such as a fetus, instructions for dosage andadministration to a patient for endothelial cell growth inhibitionand/or instructions for dosage and administration to a patient sufferingfrom hematological proliferative disorders of P antigen positive cells,e.g., Polycythemia Vera.

[0133] Some kit embodiments also contain devices for letting blood(e.g., needles and syringe, finger prick lances, cappillary tube prickdevices) and devices for low speed centrifugation of blood cells so asto enable a rapid determination of red blood cell hematocrit. Byfollowing red blood cell hematocrit, for example, one can rapidlydetermine the progress of treatment with the compositions describedherein and one can adjust the dosage or type of medicament used inresponse to hematocrit levels.

[0134] Although the invention has been described with reference toembodiments and examples, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims. All references cited herein are hereby expressly incorporated byreference.

1 63 1 7 PRT Artificial Sequence Peptide fragments derived fromparvovirus capsid particles 1 Lys Tyr Val Thr Gly Ile Asn 1 5 2 21 PRTArtificial Sequence Peptide fragments derived from parvovirus capsidparticles 2 Gly Leu Asn Met His Thr Tyr Phe Pro Asn Lys Gly Thr Gln GlnTyr 1 5 10 15 Thr Asp Gln Ile Glu 20 3 16 PRT Artificial SequencePeptide fragments derived from parvovirus capsid particles 3 Thr Tyr PhePro Asn Lys Gly Thr Gln Gln Tyr Thr Asp Gln Ile Glu 1 5 10 15 4 12 PRTArtificial Sequence Peptide fragments derived from parvovirus capsidparticles 4 Asn Lys Gly Thr Gln Gln Tyr Thr Asp Gln Ile Glu 1 5 10 5 10PRT Artificial Sequence Peptide fragments derived from parvovirus capsidparticles 5 Asn Lys Gly Thr Gln Gln Tyr Thr Asp Gln 1 5 10 6 8 PRTArtificial Sequence Peptide fragments derived from parvovirus capsidparticles 6 Asn Lys Gly Thr Gln Gln Tyr Thr 1 5 7 6 PRT ArtificialSequence Peptide fragments derived from parvovirus capsid particles 7Gln Gln Tyr Thr Asp Gln 1 5 8 4 PRT Artificial Sequence Peptidefragments derived from parvovirus capsid particles 8 Gln Gln Tyr Gln 1 920 PRT Artificial Sequence Peptide fragments derived from parvoviruscapsid 9 Met Thr Ser Val Asn Ser Ala Glu Ala Ser Thr Gly Ala Gly Gly Gly1 5 10 15 Gly Ser Asn Pro 20 10 20 PRT Artificial Sequence Peptidefragments derived from parvovirus capsid 10 Thr Gly Ala Gly Gly Gly GlySer Asn Pro Val Lys Ser Met Trp Ser 1 5 10 15 Glu Gly Ala Thr 20 11 20PRT Artificial Sequence Peptide fragments derived from parvovirus capsid11 Val Lys Ser Met Trp Ser Glu Gly Ala Thr Phe Ser Ala Asn Ser Val 1 510 15 Thr Cys Thr Phe 20 12 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 12 Phe Ser Ala Asn Ser Val Thr Cys ThrPhe Ser Arg Gln Phe Leu Ile 1 5 10 15 Pro Tyr Asp Pro 20 13 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 13Ser Arg Gln Phe Leu Ile Pro Tyr Asp Pro Glu His His Tyr Lys Val 1 5 1015 Phe Ser Pro Ala 20 14 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 14 Glu His His Tyr Lys Val Phe Ser ProAla Ala Ser Ser Cys His Asn 1 5 10 15 Ala Ser Gly Lys 20 15 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 15Ala Ser Ser Cys His Asn Ala Ser Gly Lys Glu Ala Lys Val Cys Thr 1 5 1015 Ile Ser Pro Ile 20 16 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 16 Glu Ala Lys Val Cys Thr Ile Ser ProIle Met Gly Tyr Ser Thr Pro 1 5 10 15 Trp Arg Tyr Leu 20 17 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 17Met Gly Tyr Ser Thr Pro Trp Arg Tyr Leu Asp Phe Asn Ala Leu Asn 1 5 1015 Leu Phe Phe Ser 20 18 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 18 Asp Phe Asn Ala Leu Asn Leu Phe PheSer Pro Leu Glu Phe Gln His 1 5 10 15 Leu Ile Glu Asn 20 19 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 19Pro Leu Glu Phe Gln His Leu Ile Glu Asn Tyr Gly Ser Ile Ala Pro 1 5 1015 Asp Ala Leu Thr 20 20 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 20 Tyr Gly Ser Ile Ala Pro Asp Ala LeuThr Val Thr Ile Ser Glu Ile 1 5 10 15 Ala Val Lys Asp 20 21 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 21Val Thr Ile Ser Glu Ile Ala Val Lys Asp Val Thr Asp Lys Thr Gly 1 5 1015 Gly Gly Val Gln 20 22 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 22 Val Thr Asp Lys Thr Gly Gly Gly ValGln Val Thr Asp Ser Thr Thr 1 5 10 15 Gly Arg Leu Cys 20 23 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 23Val Thr Asp Ser Thr Thr Gly Arg Leu Cys Met Leu Val Asp His Glu 1 5 1015 Tyr Lys Tyr Pro 20 24 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 24 Met Leu Val Asp His Glu Tyr Lys TyrPro Tyr Val Leu Gly Gln Gly 1 5 10 15 Gln Asp Thr Leu 20 25 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 25Tyr Val Leu Gly Gln Gly Gln Asp Thr Leu Ala Pro Glu Leu Pro Ile 1 5 1015 Trp Val Tyr Phe 20 26 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 26 Ala Pro Glu Leu Pro Ile Trp Val TyrPhe Pro Pro Gln Tyr Ala Tyr 1 5 10 15 Leu Thr Val Gly 20 27 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 27Pro Pro Gln Tyr Ala Tyr Leu Thr Val Gly Asp Val Asn Thr Gln Gly 1 5 1015 Ile Ser Gly Asp 20 28 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 28 Asp Val Asn Thr Gln Gly Ile Ser GlyAsp Ser Lys Lys Leu Ala Ser 1 5 10 15 Glu Glu Ser Ala 20 29 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 29Ser Lys Lys Leu Ala Ser Glu Glu Ser Ala Phe Tyr Val Leu Glu His 1 5 1015 Ser Ser Phe Gln 20 30 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 30 Phe Tyr Val Leu Glu His Ser Ser PheGln Leu Leu Gly Thr Gly Gly 1 5 10 15 Thr Ala Thr Met 20 31 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 31Leu Leu Gly Thr Gly Gly Thr Ala Thr Met Ser Tyr Lys Phe Pro Pro 1 5 1015 Val Pro Pro Glu 20 32 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 32 Ser Tyr Lys Phe Pro Pro Val Pro ProGlu Asn Leu Glu Gly Cys Ser 1 5 10 15 Gln His Phe Tyr 20 33 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 33Asn Leu Glu Gly Cys Ser Gln His Phe Tyr Glu Met Tyr Asn Pro Leu 1 5 1015 Tyr Gly Ser Arg 20 34 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 34 Glu Met Tyr Asn Pro Leu Tyr Gly SerArg Leu Gly Val Pro Asp Thr 1 5 10 15 Leu Gly Gly Asp 20 35 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 35Leu Gly Val Pro Asp Thr Leu Gly Gly Asp Pro Lys Phe Arg Ser Leu 1 5 1015 Thr His Glu Asp 20 36 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 36 Pro Lys Phe Arg Ser Leu Thr His GluAsp His Ala Ile Gln Pro Gln 1 5 10 15 Asn Phe Met Pro 20 37 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 37His Ala Ile Gln Pro Gln Asn Phe Met Pro Gly Pro Leu Val Asn Ser 1 5 1015 Val Ser Thr Lys 20 38 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 38 Gly Pro Leu Val Asn Ser Val Ser ThrLys Glu Gly Asp Ser Ser Asn 1 5 10 15 Thr Gly Ala Gly 20 39 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 39Glu Gly Asp Ser Ser Asn Thr Gly Ala Gly Lys Ala Leu Thr Gly Leu 1 5 1015 Ser Thr Gly Thr 20 40 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 40 Lys Ala Leu Thr Gly Leu Ser Thr GlyThr Ser Gln Asn Thr Arg Ile 1 5 10 15 Ser Leu Arg Pro 20 41 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 41Ser Gln Asn Thr Arg Ile Ser Leu Arg Pro Gly Pro Val Ser Gln Pro 1 5 1015 Tyr His His Trp 20 42 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 42 Gly Pro Val Ser Gln Pro Tyr His HisTrp Asp Thr Asp Lys Tyr Val 1 5 10 15 Thr Gly Ile Asn 20 43 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 43Asp Thr Asp Lys Tyr Val Thr Gly Ile Asn Ala Ile Ser His Gly Gln 1 5 1015 Thr Thr Tyr Gly 20 44 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 44 Ala Ile Ser His Gly Gln Thr Thr TyrGly Asn Ala Glu Asp Lys Glu 1 5 10 15 Tyr Gln Gln Gly 20 45 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 45Asn Ala Glu Asp Lys Glu Tyr Gln Gln Gly Val Gly Arg Phe Pro Asn 1 5 1015 Glu Lys Glu Gln 20 46 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 46 Val Gly Arg Phe Pro Asn Glu Lys GluGln Leu Lys Gln Leu Gln Gly 1 5 10 15 Leu Asn Met His 20 47 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 47Leu Lys Gln Leu Gln Gly Leu Asn Met His Thr Tyr Phe Pro Asn Lys 1 5 1015 Gly Thr Gln Gln 20 48 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 48 Thr Tyr Phe Pro Asn Lys Gly Thr GlnGln Tyr Thr Asp Gln Ile Glu 1 5 10 15 Arg Pro Leu Met 20 49 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 49Tyr Thr Asp Gln Ile Glu Arg Pro Leu Met Val Gly Ser Val Trp Asn 1 5 1015 Arg Arg Ala Leu 20 50 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 50 Val Gly Ser Val Trp Asn Arg Arg AlaLeu His Tyr Glu Ser Gln Leu 1 5 10 15 Trp Ser Lys Ile 20 51 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 51His Tyr Glu Ser Gln Leu Trp Ser Lys Ile Pro Asn Leu Asp Asp Ser 1 5 1015 Phe Lys Thr Gln 20 52 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 52 Pro Asn Leu Asp Asp Ser Phe Lys ThrGln Phe Ala Ala Leu Gly Gly 1 5 10 15 Trp Gly Leu His 20 53 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 53Phe Ala Ala Leu Gly Gly Trp Gly Leu His Gln Pro Pro Pro Gln Ile 1 5 1015 Phe Leu Lys Ile 20 54 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 54 Gln Pro Pro Pro Gln Ile Phe Leu LysIle Leu Pro Gln Ser Gly Pro 1 5 10 15 Ile Gly Gly Ile 20 55 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 55Leu Pro Gln Ser Gly Pro Ile Gly Gly Ile Lys Ser Met Gly Ile Thr 1 5 1015 Thr Leu Val Gln 20 56 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 56 Lys Ser Met Gly Ile Thr Thr Leu ValGln Tyr Ala Val Gly Ile Met 1 5 10 15 Thr Val Thr Met 20 57 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 57Tyr Ala Val Gly Ile Met Thr Val Thr Met Thr Phe Lys Leu Gly Pro 1 5 1015 Arg Lys Ala Thr 20 58 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 58 Thr Phe Lys Leu Gly Pro Arg Lys AlaThr Gly Arg Trp Asn Pro Gln 1 5 10 15 Pro Gly Val Tyr 20 59 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 59Gly Arg Trp Asn Pro Gln Pro Gly Val Tyr Pro Pro His Ala Ala Gly 1 5 1015 His Leu Pro Tyr 20 60 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 60 Pro Pro His Ala Ala Gly His Leu ProTyr Val Leu Tyr Asp Pro Thr 1 5 10 15 Ala Thr Asp Ala 20 61 20 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 61Val Leu Tyr Asp Pro Thr Ala Thr Asp Ala Lys Gln His His Arg His 1 5 1015 Gly Tyr Glu Lys 20 62 20 PRT Artificial Sequence Peptide fragmentsderived from parvovirus capsid 62 Lys Gln His His Arg His Gly Tyr GluLys Pro Glu Glu Leu Trp Thr 1 5 10 15 Ala Lys Ser Arg 20 63 14 PRTArtificial Sequence Peptide fragments derived from parvovirus capsid 63Pro Glu Glu Leu Trp Thr Ala Lys Ser Arg Val His Pro Leu 1 5 10

What is claimed is: 1 A method of inhibiting the growth of hematopoieticcells comprising: contacting a plurality of hematopoietic cells with agrowth inhibiting amount of a capsid agent selected from the groupconsisting of a recombinant B19 parvovirus capsid, a recombinant B19parvovirus VP2 capsid, and a fragment of a recombinant B19 parvovirusVP2 capsid, wherein said fragment is at least three amino acids inlength; and measuring the inhibition of growth of said hematopoieticcells.
 2. The method of claim 1, wherein a growth inhibiting amount ofthe recombinant B19 parvovirus capsid is contacted with said pluralityof hematopoietic cells.
 3. The method of claim 1, wherein a growthinhibiting amount of the recombinant B19 parvovirus VP2 capsid iscontacted with said plurality of hematopoietic cells.
 4. The method ofclaim 1, wherein a growth inhibiting amount of the fragment of arecombinant B19 parvovirus VP2 capsid is contacted with said pluralityof hematopoietic cells.
 5. The method of claim 4, wherein said fragmentconsists of the sequence glutamine-glutamine-tyrosine.
 6. The method ofclaim 4, wherein said fragment consists of the sequence of SEQ. ID. No.5.
 7. The method of claim 1, wherein said measuring step comprisesobserving a reduction in the presence of a hematopoietic cell.
 8. Themethod of claim 1, wherein said measuring step involves observing areduction in red blood cell hematocrit. 9 A method of inhibiting theproliferation of endothelial cells comprising: contacting a plurality ofendothelial cells with a proliferation inhibiting amount of a capsidagent selected from the group consisting of a recombinant B19 parvoviruscapsid and a recombinant B19 parvovirus VP2 capsid; and measuring theinhibition of proliferation of said endothelial cells.
 10. The method ofclaim 9, wherein a proliferation inhibiting amount of the recombinantB19 parvovirus capsid is contacted with said plurality of endothelialcells.
 11. The method of claim 9, wherein a proliferation inhibitingamount of the recombinant B19 parvovirus VP2 capsid is contacted withsaid plurality of endothelial cells.
 12. The method of claim 9, whereinsaid measuring step comprises observing a reduction in the presence ofan endothelial cell. 13 A method of inhibiting the migration ofendothelial cells comprising: contacting a plurality of endothelialcells with a migration inhibiting amount of a capsid agent selected fromthe group consisting of a recombinant B19 parvovirus capsid, arecombinant B19 parvovirus VP1 capsid, and a recombinant B19 parvovirusVP2 capsid; and measuring the inhibition of migration of saidendothelial cells.
 14. The method of claim 13, wherein a migrationinhibiting amount of the recombinant B19 parvovirus capsid is contactedwith said plurality of endothelial cells.
 15. The method of claim 13,wherein a migration inhibiting amount of the recombinant B19 parvovirusVP1 capsid is contacted with said plurality of endothelial cells. 16.The method of claim 13, wherein a migration inhibiting amount of therecombinant B19 parvovirus VP2 capsid is contacted with said pluralityof endothelial cells.
 17. The method of claim 13, wherein said measuringstep involves observing a reduction in metastasis or angiogenesis. 18 Amethod of inhibiting the growth of hematopoietic cells comprising:identifying a subject in need of an inhibition of growth ofhematopoietic cells; and providing to said subject a growth inhibitingamount of a capsid agent selected from the group consisting of arecombinant B19 parvovirus capsid, a recombinant B19 parvovirus VP2capsid, and a fragment of a recombinant B19 parvovirus VP2 capsid,wherein said fragment is at least three amino acids in length.
 19. Themethod of claim 18, wherein a growth inhibiting amount of therecombinant B19 parvovirus capsid is provided to said subject.
 20. Themethod of claim 18, wherein a growth inhibiting amount of therecombinant B19 parvovirus VP2 capsid is provided to said subject. 21.The method of claim 18, wherein a growth inhibiting amount of thefragment of a recombinant B19 parvovirus VP2 capsid is provided to saidsubject.
 22. The method of claim 18, wherein said fragment consists ofthe sequence glutamine-glutamine-tyrosine.
 23. The method of claim 18,wherein said fragment consists of the sequence of SEQ. ID. No.5.
 24. Themethod of claim 18, wherein said subject has a hematologicalproliferative disorder.
 25. The method of claim 24, wherein saidhematological proliferative disorder is Polycythemia Vera. 26 A methodof inhibiting the proliferation of endothelial cells comprising:identifying a subject in need of an inhibition of proliferation ofendothelial cells; and providing to said subject a proliferationinhibiting amount of a capsid agent selected from the group consistingof a recombinant B19 parvovirus capsid and a recombinant B19 parvovirusVP2 capsid.
 27. The method of claim 26, wherein a proliferationinhibiting amount of the recombinant B19 parvovirus capsid is providedto said subject.
 28. The method of claim 26, wherein a proliferationinhibiting amount of the recombinant B19 parvovirus VP2 capsid isprovided to said subject. 29 A method of inhibiting the migration ofendothelial cells comprising: identifying a subject in need of aninhibition of migration of endothelial cells; and providing to saidsubject a migration inhibiting amount of a capsid agent selected fromthe group consisting of a recombinant B19 parvovirus capsid, arecombinant B19 parvovirus VP1 capsid, and a recombinant B19 parvovirusVP2 capsid.
 30. The method of claim 29, wherein a migration inhibitingamount of the recombinant B19 parvovirus capsid is provided to saidsubject.
 31. The method of claim 29, wherein a migration inhibitingamount of the recombinant B19 parvovirus VP1 capsid is provided to saidsubject.
 32. The method of claim 29, wherein a migration inhibitingamount of the recombinant B19 parvovirus VP2 capsid is provided to saidsubject.
 33. An isolated or purified fragment of parvovirus B19 VP2capsid consisting of a sequence selected from the group consisting ofglutamine-glutamine-tyrosine, SEQ. ID. NO: 5, SEQ. ID. NO: 6, SEQ. ID.NO: 7, SEQ. ID. NO: 8, SEQ. ID. NO: 44, SEQ. ID. NO: 45, SEQ. ID. NO:46, SEQ. ID. NO: 47, SEQ. ID. NO: 48, SEQ. ID. NO: 49, and SEQ. ID. NO:50.